Methods and reagents for diagnosis and treatment of diabetes

ABSTRACT

The invention relates to methods and reagents for diagnosing and treating diabetes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/308,393, filed Dec. 2, 2002, which claims priority to U.S.Provisional Patent Application Ser. No. 60/336,633, filed on Dec. 3,2001, each of which is incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to methods and reagents useful for treatment anddiagnosis of diabetes.

BACKGROUND OF THE INVENTION

Diabetes mellitus can be divided into two clinical syndromes, Type 1 andType 2 diabetes mellitus. Type 1, or insulin-dependent diabetes mellitus(IDDM), is a chronic autoimmune disease characterized by the extensiveloss of beta cells in the pancreatic Islets of Langerhans (hereinafterreferred to as “pancreatic islet cells” or “islet cells”), which produceinsulin. As these cells are progressively destroyed, the amount ofsecreted insulin decreases, eventually leading to hyperglycemia(abnormally high level of glucose in the blood) when the amount secreteddrops below the level required for euglycemia (normal blood glucoselevel). Although the exact trigger for this immune response is notknown, patients with IDDM have high levels of antibodies againstpancreatic beta cells. However, not all patients with high levels ofthese antibodies develop IDDM.

Type 2 diabetes develops when muscle, fat and liver cells fail torespond normally to insulin. This failure to respond (called insulinresistance) may be due to reduced numbers of insulin receptors on thesecells, or a dysfunction of signaling pathways within the cells, or both.The beta cells initially compensate for this insulin resistance byincreasing their insulin output. Over time, these cells become unable toproduce enough insulin to maintain normal glucose levels, indicatingprogression to type 2 diabetes.

Type 2 diabetes is brought on by a combination of poorly understoodgenetic and acquired risk factors—including a high-fat diet, lack ofexercise, and aging. Worldwide, type 2 diabetes has become an epidemic,driven by increases in obesity and a sedentary lifestyle, widespreadadoption of western dietary habits, and the general aging of thepopulations in many countries. In 1985, an estimated 30 million peopleworldwide had diabetes—by 2000, this figure had increased 5-fold, to anestimated 154 million people. The number of people with diabetes isexpected double between now and 2025, to about 300 million.

There is no cure for diabetes. Conventional treatments for diabetes arevery limited, and focus on attempting to control blood glucose levels inorder to minimize or delay complications. The present inventionaddresses these and other problems.

BRIEF SUMMARY OF THE INVENTION

This invention provides isolated nucleic acids encoding an Archipelinpolypeptide. In some embodiments, the Archipelin polypeptide is at least60% identical to SEQ ID NO:2. In some embodiments, the nucleic acidencodes SEQ ID NO:2. In some embodiments, wherein the nucleic acidcomprises SEQ ID NO: 1. In some embodiments, the nucleic acid encodesSEQ ID NO:7. In some embodiments, the nucleic acid comprises SEQ IDNO:8. In some embodiments, the nucleic acid encodes SEQ ID NO:6. In someembodiments, the nucleic acid comprises SEQ ID NO:5. In someembodiments, the polypeptide comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14. In some embodiments, thenucleic acid is amplified by a primer set selected from the groupconsisting of GCGATGTTGAAGAAGAAGTTC (SEQ ID NO: 15) and ATCGCCAAGGCCAAGA(SEQ ID NO:16).

The present invention also provides expression cassettes comprising apromoter operably linked to a nucleic acid encoding an polypeptide atleast 605 identical to SEQ ID NO:9.

The present invention also provides isolated nucleic acids thatspecifically hybridizes following at least one wash in 0.2X SSC at 55°C. for 20 minutes to a probe comprising SEQ ID NO: 1.

The present invention also provides isolated polypeptides comprising anamino acid sequence at least 60% identical to SEQ ID NO:9. In someembodiments, the polypeptide comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11,SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14. In some embodiments,the polypeptide comprises SEQ ID NO:2. In some embodiments, thepolypeptide comprises SEQ ID NO:6. In some embodiments, the polypeptidespecifically binds to antibodies generated against SEQ ID NO:9.

The present invention also provides antibodies that specificallyhybridizes to Archipelin polypeptides. In some embodiments, polypeptidescomprising an amino acid sequence at least 60% identical to SEQ ID NO:9.

The present invention also provides host cells transfected with anucleic acid encoding an polypeptide at least 60% identical to SEQ IDNO:9. In some embodiments, the cell is a pancreatic islet cell.

The present invention also provides pharmaceutical compositionscomprising insulin and a polypeptide at least 60% identical to SEQ IDNO:9. In some embodiments, the polypeptide comprises an amino acidsequence selected from the group consisting of SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14. Insome embodiments, the polypeptide comprises SEQ ID NO:2. In someembodiments, the pharmaceutical composition is suitable for injection.

The present invention also provides methods of diagnosing type 1 or type2 diabetes or a predisposition for type 1 or type 2 diabetes in apatient. In some embodiments, the methods comprise detecting the levelof a polypeptide at least 60% identical to SEQ ID NO:9 in a sample fromthe patient, wherein a modulated level of the polypeptide in the samplecompared to a level of the polypeptide in a non-diabetic individualindicates that the patient is diabetic or is predisposed for at leastsome pathological aspects of diabetes. In some embodiments, themodulated level of the polypeptide in the sample is lower than a levelof the polypeptide in a non-diabetic individual. In some embodiments,the modulated level of the polypeptide in the sample is higher than alevel of the polypeptide in a non-diabetic individual. In someembodiments, the polypeptide comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14. In some embodiments, thepolypeptide comprises SEQ ID NO:2. In some embodiments, the polypeptideis detected by an antibody. In some embodiments, the level of thepolypeptide in the patient is less than 50% of the level from thenon-diabetic individual. In some embodiments, the level of thepolypeptide in the patient is at least 150% of the level from thenon-diabetic individual.

The present invention also provides methods of treating a patientdiagnosed with type 1 or type 2 diabetes. In some embodiments, themethod comprise administering to the patient a pharmaceuticalcomposition comprising a therapeutically effective amount of a compoundselected from the group consisting of an agonist of Archipelin and anagent that increases expression of Archipelin. In some embodiments, thecompound is an agonist of Archipelin. In some embodiments, the compoundis an agent that increases expression of Archipelin. In someembodiments, the agonist is a polypeptide at least 60% identical to SEQID NO:9. In some embodiments, the agonist comprises a polypeptidecomprising SEQ ID NO:2. In some embodiments, the polypeptide comprisesan amino acid sequence selected from the group consisting of SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ IDNO:14. In some embodiments, the pharmaceutical composition comprisesinsulin. In some embodiments, the pharmaceutical composition isadministered parenterally. In some embodiments, the pharmaceuticalcomposition is administered by injection. In some embodiments, thepharmaceutical composition is administered by a pump device.

The present invention also provides methods of modulating Archipelinactivity in a cell. In some embodiments, the methods compriseintroducing into a pancreatic islet cell an expression cassettecomprising a promoter operably linked to a polynucleotide encoding apolypeptide at least 60% identical to SEQ ID NO:9. In some embodiments,the polypeptide comprises SEQ ID NO:2. In some embodiments, thepolypeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO:12,SEQ ID NO:13, and SEQ ID NO:14. In some embodiments, the cell isintroduced into a patient. In some embodiments, the cell is from thepatient. In some embodiments, the expression cassette is contained in aviral vector.

The present invention also provides methods of identifying an agentuseful for the treatment of diabetes. In some embodiments, the methodscomprise contacting a cell with an agent; and selecting an agent thatmodulates the expression in the cell of a polypeptide at least 60%identical to SEQ ID NO:9. In some embodiments, the polypeptide comprisesan amino acid sequence selected from the group consisting of SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ IDNO:14. In some embodiments, the cell is from a diabetic animal. In someembodiments, the diabetic animal is a human. In some embodiments, thecell is a pancreatic islet cell. In some embodiments, the polypeptidecomprises SEQ ID NO:2. In some embodiments, the expression of thepolypeptide is increased following the contacting step.

The present invention also provides methods of treating a patientdiagnosed with type 1 or type 2 diabetes. In some embodiments, themethods comprise administering a therapeutically effective amount of anagent which was identified by contacting a cell with an agent; andselecting an agent that modulates the expression in the cell of apolypeptide at least 60% identical to SEQ ID NO:9. In some embodiments,the agent increases the expression of the polypeptide in the patient.

DEFINITIONS

“Antibody” refers to a polypeptide substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof whichspecifically bind and recognize an analyte (antigen). The recognizedimmunoglobulin genes include the kappa, lambda, alpha, gamma, delta,epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′₂, a dimer of Fab whichitself is a light chain joined to V_(H)-C_(H) 1 by a disulfide bond. TheF(ab)′₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region, thereby converting the F(ab)′₂ dimer intoan Fab′ monomer. The Fab′ monomer is essentially an Fab with part of thehinge region (see, Paul (Ed.) Fundamental Immunology, Third Edition,Raven Press, NY (1993)). While various antibody fragments are defined interms of the digestion of an intact antibody, one of skill willappreciate that such fragments may be synthesized de novo eitherchemically or by utilizing recombinant DNA methodology. Thus, the termantibody, as used herein, also includes antibody fragments eitherproduced by the modification of whole antibodies or those synthesized denovo using recombinant DNA methodologies (e.g., single chain Fv).

The terms “peptidomimetic” and “mimetic” refer to a synthetic chemicalcompound that has substantially the same structural and functionalcharacteristics of the Archipelin polypeptides of the invention. Peptideanalogs are commonly used in the pharmaceutical industry as non-peptidedrugs with properties analogous to those of the template peptide. Thesetypes of non-peptide compound are termed “peptide mimetics” or“peptidomimetics” (Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber andFreidinger TINS p. 392 (1985); and Evans et al. J Med. Chem. 30:1229(1987), which are incorporated herein by reference). Peptide mimeticsthat are structurally similar to therapeutically useful peptides may beused to produce an equivalent or enhanced therapeutic or prophylacticeffect. Generally, peptidomimetics are structurally similar to aparadigm polypeptide (i.e., a polypeptide that has a biological orpharmacological activity), such as found in Archipelin, but have one ormore peptide linkages optionally replaced by a linkage selected from thegroup consisting of, e.g., —CH2NH—, —CH2S—, —CH2—CH2—, —CH═CH—(cis andtrans), —COCH2—, —CH(OH)CH2—, and —CH2SO—. The mimetic can be eitherentirely composed of synthetic, non-natural analogues of amino acids,or, is a chimeric molecule of partly natural peptide amino acids andpartly non-natural analogs of amino acids. The mimetic can alsoincorporate any amount of natural amino acid conservative substitutionsas long as such substitutions also do not substantially alter themimetic's structure and/or activity. For example, a mimetic compositionis within the scope of the invention if it is capable of carrying outthe binding or enzymatic activities of Archipelin .

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

The term “isolated,” when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is essentially free of other cellularcomponents with which it is associated in the natural state. It ispreferably in a homogeneous state although it can be in either a dry oraqueous solution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinwhich is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames which flank the gene and encode a protein otherthan the gene of interest. The term “purified” denotes that a nucleicacid or protein gives rise to essentially one band in an electrophoreticgel. Particularly, it means that the nucleic acid or protein is at least85% pure, more preferably at least 95% pure, and most preferably atleast 99% pure.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides whichhave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes.19:5081 (1991); Ohtsuka et al., J Biol. Chem. 260:2605-2608 (1985); andCassol et al. (1992); Rossolini et al., Mol. Cell. Probes 8:91-98(1994)). The term nucleic acid is used interchangeably with gene, cDNA,and mRNA encoded by a gene.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers. As usedherein, the terms encompass amino acid chains of any length, includingfull length proteins (i.e., antigens), wherein the amino acid residuesare linked by covalent peptide bonds.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. “Amino acid mimetics” refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

An “Archipelin nucleic acid” or “Archipelin polynucleotide sequence” ofthe invention is a subsequence or full-length polynucleotide sequence ofa gene that encodes a polypeptide expressed in pancreatic islet cells.Exemplary Archipelin nucleic acids of the invention include sequencessubstantially identical to SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, andSEQ ID NO:8. Similarly, “Archipelin polypeptide” or “Archipelin” refersto a polypeptide, or fragment thereof, that is substantially identicalto a polypeptide encoded by SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, andSEQ ID NO:8 (e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7,and SEQ ID NOs:9-14) or peptidomimetic compositions with substantiallythe same activity as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7,and SEQ ID NOs:9-14.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

-   1) Alanine (A), Glycine (G);-   2) Aspartic acid (D), Glutamic acid (E);-   3) Asparagine (N), Glutamine (Q);-   4) Arginine (R), Lysine (K);-   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);-   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);-   7) Serine (S), Threonine (T); and-   8) Cysteine (C), Methionine (M)-   (see, e.g., Creighton, Proteins (1984)).

Macromolecular structures such as polypeptide structures can bedescribed in terms of various levels of organization. For a generaldiscussion of this organization, see, e.g., Alberts et al., MolecularBiology of the Cell (3^(rd) ed., 1994) and Cantor and Schimmel,Biophysical Chemistry Part I. The Conformation of BiologicalMacromolecules (1980). “Primary structure” refers to the amino acidsequence of a particular peptide. “Secondary structure” refers tolocally ordered, three dimensional structures within a polypeptide.These structures are commonly known as domains. Domains are portions ofa polypeptide that form a compact unit of the polypeptide and aretypically 50 to 350 amino acids long. Typical domains are made up ofsections of lesser organization such as stretches of β-sheet andα-helices. “Tertiary structure” refers to the complete three dimensionalstructure of a polypeptide monomer. “Quaternary structure” refers to thethree dimensional structure formed by the noncovalent association ofindependent tertiary units. Anisotropic terms are also known as energyterms.

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95%identity over a specified region), when compared and aligned for maximumcorrespondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. Such sequences are then said tobe “substantially identical.” This definition also refers to thecomplement of a test sequence. Optionally, the identity exists over aregion that is at least about 50 amino acids or nucleotides in length,or more preferably over a region that is 75-100 amino acids ornucleotides in length.

The term “similarity,” or percent “similarity,” in the context of two ormore polypeptide sequences, refer to two or more sequences orsubsequences that have a specified percentage of amino acid residuesthat are either the same or similar as defined in the 8 conservativeamino acid substitutions defined above (i.e., 60%, optionally 65%, 70%,75%, 80%, 85%, 90%, or 95% similar over a specified region), whencompared and aligned for maximum correspondence over a comparisonwindow, or designated region as measured using one of the followingsequence comparison algorithms or by manual alignment and visualinspection. Such sequences are then said to be “substantially similar.”Optionally, this identity exists over a region that is at least about 50amino acids in length, or more preferably over a region that is at leastabout 75-100 amino acids in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homologyalignment algorithm of Needleman and Wunsch (1970) J Mol. Biol. 48:443,by the search for similarity method of Pearson and Lipman (1988) Proc.Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,WI), or by manual alignment and visual inspection (see, e.g., Ausubel etal., Current Protocols in Molecular Biology (1995 supplement)).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng and Doolittle (1987) J Mol. Evol.35:351-360. The method used is similar to the method described byHiggins and Sharp (1989) CABIOS 5:151-153. The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package, e.g., version 7.0 (Devereaux etal. (1984) Nuc. Acids Res. 12:387-395).

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1977) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (e.g., total cellular orlibrary DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength pH. The T_(m) is the temperature (under definedionic strength, pH, and nucleic concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at T_(m),50% of the probes are occupied at equilibrium). Stringent conditionswill be those in which the salt concentration is less than about 1.0 Msodium ion, typically about 0.01 to 1.0 M sodium ion concentration (orother salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C. for long probes (e.g., greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide. For selective or specific hybridization, apositive signal is at least two times background, optionally 10 timesbackground hybridization. Exemplary stringent hybridization conditionscan be as following: 50% formamide, 5X SSC, and 1% SDS, incubating at42° C., or 5X SSC, 1% SDS, incubating at 65° C., with wash in 0.2X SSC,and 0.1% SDS at 65° C. Such washes can be performed for 5, 15, 30, 60,120, or more minutes.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1X SSC at 45° C. Such washes can be performed for 5, 15,30, 60, 120, or more minutes. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency.

The phrase “a nucleic acid sequence encoding” refers to a nucleic acidwhich contains sequence information for a structural RNA such as rRNA, atRNA, or the primary amino acid sequence of a specific protein orpeptide, or a binding site for a trans-acting regulatory agent. Thisphrase specifically encompasses degenerate codons (i.e., differentcodons which encode a single amino acid) of the native sequence orsequences which may be introduced to conform with codon preference in aspecific host cell.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (nonrecombinant) form of the cell or expressnative genes that are otherwise abnormally expressed, under-expressed ornot expressed at all.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation. The term“operably linked” refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the expression control sequence directs transcription of thenucleic acid corresponding to the second sequence.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

The phrase “specifically (or selectively) binds to an antibody” or“specifically (or selectively) immunoreactive with”, when referring to aprotein or peptide, refers to a binding reaction which is determinativeof the presence of the protein in the presence of a heterogeneouspopulation of proteins and other biologics. Thus, under designatedimmunoassay conditions, the specified antibodies bind to a particularprotein and do not bind in a significant amount to other proteinspresent in the sample. Specific binding to an antibody under suchconditions may require an antibody that is selected for its specificityfor a particular protein. For example, antibodies raised against aprotein having an amino acid sequence encoded by any of thepolynucleotides of the invention can be selected to obtain antibodiesspecifically immunoreactive with that protein and not with otherproteins, except for polymorphic variants. A variety of immunoassayformats may be used to select antibodies specifically immunoreactivewith a particular protein. For example, solid-phase ELISA immunoassays,Western blots, or immunohistochemistry are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. See,Harlow and Lane Antibodies, A Laboratory Manual, Cold Spring HarborPublications, NY (1988) for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity.Typically, a specific or selective reaction will be at least twice thebackground signal or noise and more typically more than 10 to 100 timesbackground.

“Inhibitors,” “activators,” and “modulators” of Archipelin expression orof Archipelin activity are used to refer to inhibitory, activating, ormodulating molecules, respectively, identified using in vitro and invivo assays for Archipelin expression or Archipelin signaling, e.g.,ligands, agonists, antagonists, and their homologs and mimetics.Modulators include inhibitors and activators. Inhibitors are compoundsthat, e.g., inhibit expression of Archipelin or bind to, partially ortotally block stimulation, decrease, prevent, delay activation,inactivate, desensitize, or down regulate the activity of Archipelin orthat bind or down regulate a receptor to which Archipelin binds. e.g.,antagonists. Activators are compounds that, e.g., induce or activate theexpression of a Archipelin or bind to, stimulate, increase, open,activate, facilitate, enhance activation, sensitize or up regulate theactivity of Archipelin or that bind or up regulate a receptor to whichArchipelin binds, e.g., agonists. Modulators include compounds that,e.g., alter the interaction of Archipelin with: extracellular proteinsthat bind activators or inhibitors, receptors, including G-proteinscoupled-receptors (GPCRs), kinases, etc. Modulators include geneticallymodified versions of Archipelin, e.g., with altered activity, as well asnaturally occurring and synthetic ligands, antagonists, agonists, smallchemical molecules and the like. Such assays for inhibitors andactivators include, e.g., applying putative modulator compounds topancreatic islet cells, in the presence or absence of Archipelin andthen determining the functional effects on Archipelin signaling, asdescribed above. Samples or assays comprising Archipelin that aretreated with a potential activator, inhibitor, or modulator are comparedto control samples without the inhibitor, activator, or modulator toexamine the extent of inhibition. Control samples (untreated withinhibitors) are assigned a relative Archipelin activity value of 100%.Inhibition of a Archipelin is achieved when the Archipelin activityvalue relative to the control is less than about 80%, optionally lessthan about 50% or less than about 25-0%. Activation of a Archipelin isachieved when the Archipelin activity value relative to the control isat least about 110%, optionally at least about 150%, optionally at leastabout 200-500%, or at least about 1000-3000% or higher.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the expression pattern of probe set MBXRATISL12276 invarious tissues of rat.

FIG. 2 illustrates the expression pattern of probe set MBXRATISL12276 invarious tissues of mouse.

FIG. 3 illustrates an amino acid alignment of Archipelin sequences (SEQID NOS:33 and 34) compared to peptides from the CRF peptide family (SEQID NOS: 17-32).

FIG. 4 illustrates potential amino-terminal and carboxyl-terminalcleavage sites of the human Archipelin proprotein (SEQ ID NO:2).Potential amino terminal cleavage sites are marked with a “/”. Potentialcarboxyl terminal cleavage sites are marked with a “!” Mature humanArchipelin peptides=SEQ ID NOS:35-40.

FIG. 5 illustrates potential amino-terminal and carboxyl-terminalcleavage sites of the mouse Archipelin proprotein (SEQ ID NO:4).Potential amino terminal cleavage sites are marked with a “/”. Potentialcarboxyl terminal cleavage sites are marked with a “!”. Mature mouseArchipelin peptides=SEQ ID NOS:41-45.

FIG. 6 illustrates potential amino-terminal and carboxyl-terminalcleavage sites of the rat Archipelin proprotein (SEQ ID NO:6). Potentialamino terminal cleavage sites are marked with a “/”. Potential carboxylterminal cleavage sites are marked with a “!”. Mature rat Archipelinpeptides=SEQ ID NOS:41, 42, 46, 47, 45 and 48, respectively.

FIG. 7 illustrates detection of Archipelin in human serum using surfaceenhanced laser desorption ionization (SELDI) mass spectroscopy. [60]FIG. 8 illustrates detection of Archipelin (SEQ ID NOS:49-52) in ratserum using surface enhanced laser desorption ionization (SELDI) massspectroscopy.

DETAILED DESCRIPTION

I. Introduction

This invention is directed to new polypeptide and polynucleotidesequences, designated Archipelin sequences, as well as methods of usingthe sequences to diagnose and treat diabetes. The present method alsoprovides methods of identifying modulators of Archipelin expression andactivity. Such modulators are useful for treating type 1 and type 2diabetes as well as the pathological aspects of such diseases.

II. General Recombinant Nucleic Acids Methods for Use With the Invention

In numerous embodiments of the present invention, nucleic acids encodinga Archipelin of interest will be isolated and cloned using recombinantmethods. Such embodiments are used, e.g., to isolate Archipelinpolynucleotides (e.g., SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, and SEQID NO:8) for protein expression or during the generation of variants,derivatives, expression cassettes, or other sequences derived from anArchipelin polypeptide (e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:7, and SEQ ID NOs:9-14), to monitor Archipelin gene expression,for the isolation or detection of Archipelin sequences in differentspecies, for diagnostic purposes in a patient, e.g., to detect mutationsin Archipelin or to detect expression levels of Archipelin nucleic acidsor Archipelin polypeptides. In some embodiments, the sequences encodingthe Archipelin of the invention are operably linked to a heterologouspromoter. In one embodiment, the nucleic acids of the invention are fromany mammal, including, in particular, e.g., a human, a mouse, a rat,etc.

A. General Recombinant Nucleic Acids Methods

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 1989); Kriegler, Gene Transfer and Expression. ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from sequenced proteins, from derived amino acidsequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemicallysynthesized according to the solid phase phosphoramidite triester methodfirst described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862(1981), using an automated synthesizer, as described in Van Devanter et.al., Nucleic Acids Res. 12:6159-6168 (1984). Purification ofoligonucleotides is by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson & Reanier, J Chrom.255:137-149 (1983).

The sequence of the cloned genes and synthetic oligonucleotides can beverified after cloning using, e.g., the chain termination method forsequencing double-stranded templates of Wallace et al., Gene 16:21-26(1981).

B. Cloning Methods for the Isolation of Nucleotide Sequences Encodingthe Desired Proteins

In general, the nucleic acids encoding the subject proteins are clonedfrom DNA sequence libraries that are made to encode copy DNA (cDNA) orgenomic DNA. The particular sequences can be located by hybridizing withan oligonucleotide probe, the sequence of which can be derived from thesequences provided herein (e.g., SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5,and SEQ ID NO:8), which provides a reference for PCR primers and definessuitable regions for isolating Archipelin-specific probes.Alternatively, where the sequence is cloned into an expression library,the expressed recombinant protein can be detected immunologically withantisera or purified antibodies made against the Archipelin of interest.

Methods for making and screening genomic and cDNA libraries are wellknown to those of skill in the art (see, e.g., Gubler and Hoffman Gene25:263-269 (1983); Benton and Davis Science, 196:180-182 (1977); andSambrook, supra). A islet cells are an example of suitable cells toisolate Archipelin RNA and cDNA.

Briefly, to make the cDNA library, one should choose a source that isrich in mRNA. The mRNA can then be made into cDNA, ligated into arecombinant vector, and transfected into a recombinant host forpropagation, screening and cloning. For a genomic library, the DNA isextracted from a suitable tissue and either mechanically sheared orenzymatically digested to yield fragments of preferably about 5-100 kb.The fragments are then separated by gradient centrifugation fromundesired sizes and are constructed in bacteriophage lambda vectors.These vectors and phage are packaged in vitro, and the recombinantphages are analyzed by plaque hybridization. Colony hybridization iscarried out as generally described in Grunstein et al., Proc. Natl. AcadSci. USA., 72:3961-3965 (1975).

An alternative method combines the use of synthetic oligonucleotideprimers with polymerase extension on an mRNA or DNA template. Suitableprimers can be designed from specific Archipelin sequences, e.g., thesequences described in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, and SEQID NO:8. This polymerase chain reaction (PCR) method amplifies thenucleic acids encoding the protein of interest directly from mRNA, cDNA,genomic libraries or cDNA libraries. Restriction endonuclease sites canbe incorporated into the primers. Polymerase chain reaction or other invitro amplification methods may also be useful, for example, to clonenucleic acids encoding specific proteins and express said proteins, tosynthesize nucleic acids that will be used as probes for detecting thepresence of mRNA encoding an Archipelin polypeptide of the invention inphysiological samples, for nucleic acid sequencing, or for otherpurposes (see, U.S. Pat. Nos. 4,683,195 and 4,683,202). Genes amplifiedby a PCR reaction can be purified from agarose gels and cloned into anappropriate vector.

Appropriate primers and probes for identifying the genes encoding anArchipelin polypeptide of the invention from mammalian tissues can bederived from the sequences provided herein, in particular SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:8. For a general overview ofPCR, see, Innis et al. PCR Protocols: A Guide to Methods andApplications, Academic Press, San Diego (1990).

Synthetic oligonucleotides can be used to construct genes. This is doneusing a series of overlapping oligonucleotides, usually 40-120 bp inlength, representing both the sense and anti-sense strands of the gene.These DNA fragments are then annealed, ligated and cloned.

A gene encoding an Archipelin polypeptide of the invention can be clonedusing intermediate vectors before transformation into mammalian cellsfor expression. These intermediate vectors are typically prokaryotevectors or shuttle vectors. The proteins can be expressed in eitherprokaryotes, using standard methods well known to those of skill in theart, or eukaryotes as described infra.

C. Expression in Prokaryotes and Eukaryotes

To obtain high level expression of a cloned gene, such as cDNAs encodingArchipelin, one typically subclones polynucleotides encoding Archipelininto an expression vector that contains a strong promoter to directtranscription, a transcription/translation terminator, and if for anucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are well known inthe art and described, e.g., in Sambrook et al. and Ausubel et al.Bacterial expression systems for expressing the Archipelin protein areavailable in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al.,Gene 22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983). Kitsfor such expression systems are commercially available. Eukaryoticexpression systems for mammalian cells, yeast, and insect cells are wellknown in the art and are also commercially available.

Selection of the promoter used to direct expression of a heterologousnucleic acid depends on the particular application. The promoter ispreferably positioned about the same distance from the heterologoustranscription start site as it is from the transcription start site inits natural setting. As is known in the art, however, some variation inthis distance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of Archipelin-encodingnucleic acid in host cells. A typical expression cassette thus containsa promoter operably linked to the nucleic acid sequence encodingArchipelin and signals required for efficient polyadenylation of thetranscript, ribosome binding sites, and translation termination.Additional elements of the cassette may include enhancers and, ifgenomic DNA is used as the structural gene, introns with functionalsplice donor and acceptor sites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and fusionexpression systems such as GST and LacZ. Epitope tags can also be addedto recombinant proteins to provide convenient methods of isolation,e.g., c-myc.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A⁺,pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the CMV promoter, SV40early promoter, SV40 later promoter, metallothionein promoter, murinemammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrinpromoter, or other promoters shown effective for expression ineukaryotic cells.

Expression of proteins from eukaryotic vectors can be also be regulatedusing inducible promoters. With inducible promoters, expression levelsare tied to the concentration of inducing agents, such as tetracyclineor ecdysone, by the incorporation of response elements for these agentsinto the promoter. Generally, high level expression is obtained frominducible promoters only in the presence of the inducing agent; basalexpression levels are minimal. Inducible expression vectors are oftenchosen if expression of the protein of interest is detrimental toeukaryotic cells.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase and dihydrofolate reductase. Alternatively,high yield expression systems not involving gene amplification are alsosuitable, such as using a baculovirus vector in insect cells, with anArchipelin-encoding sequence under the direction of the polyhedrinpromoter or other strong baculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are preferably chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of Archipelinprotein, which are then purified using standard techniques (see, e.g.,Colley et al., J Biol. Chem. 264:17619-17622 (1989); Guide to ProteinPurification, in Methods in Enzymology, vol. 182 (Deutscher, ed.,1990)). Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J Bact.132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology101:347-362 (Wu et al., eds, 1983).

Any of the well-known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA or other foreign genetic material into a host cell (see,e.g., Sambrook et al., supra). It is only necessary that the particulargenetic engineering procedure used be capable of successfullyintroducing at least one gene into the host cell capable of expressingArchipelin.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofArchipelin, which is recovered from the culture using standardtechniques identified below.

III. Purification of Proteins of the Invention

Either naturally occurring or recombinant Archipelin can be purified foruse in functional assays. Naturally occurring Archipelin can bepurified, e.g., from mouse or human tissue such as islet cells or anyother source of an Archipelin ortholog. Recombinant Archipelin can bepurified from any suitable expression system.

The Archipelin may be purified to substantial purity by standardtechniques, including selective precipitation with such substances asammonium sulfate; column chromatography, immunopurification methods, andothers (see, e.g., Scopes, Protein Purification. Principles and Practice(1982); U.S. Pat. No. 4,673,641; Ausubel et al., supra; and Sambrook etal., supra).

A number of procedures can be employed when recombinant Archipelin arebeing purified. For example, proteins having established molecularadhesion properties can be reversible fused to Archipelin. With theappropriate ligand, Archipelin can be selectively adsorbed to apurification column and then freed from the column in a relatively pureform. The fused protein is then removed by enzymatic activity. FinallyArchipelin can be purified using immunoaffinity columns.

A. Purification of Proteins from Recombinant Bacteria

When recombinant proteins are expressed by the transformed bacteria inlarge amounts, typically after promoter induction, although expressioncan be constitutive, the proteins may form insoluble aggregates. Thereare several protocols that are suitable for purification of proteininclusion bodies. For example, purification of aggregate proteins(hereinafter referred to as inclusion bodies) typically involves theextraction, separation and/or purification of inclusion bodies bydisruption of bacterial cells typically, but not limited to, byincubation in a buffer of about 100-150 μg/ml lysozyme and 0.1% NonidetP40, a non-ionic detergent. The cell suspension can be ground using aPolytron grinder (Brinkman Instruments, Westbury, N.Y.). Alternatively,the cells can be sonicated on ice. Alternate methods of lysing bacteriaare described in Ausubel et al. and Sambrook et al., both supra, andwill be apparent to those of skill in the art.

The cell suspension is generally centrifuged and the pellet containingthe inclusion bodies resuspended in buffer which does not dissolve butwashes the inclusion bodies, e.g., 20 mM Tris-HCl (pH 7.2), 1 mM EDTA,150 mM NaCl and 2% Triton-X 100, a non-ionic detergent. It may benecessary to repeat the wash step to remove as much cellular debris aspossible. The remaining pellet of inclusion bodies may be resuspended inan appropriate buffer (e.g., 20 mM sodium phosphate, pH 6.8, 150 mMNaCl). Other appropriate buffers will be apparent to those of skill inthe art.

Following the washing step, the inclusion bodies are solubilized by theaddition of a solvent that is both a strong hydrogen acceptor and astrong hydrogen donor (or a combination of solvents each having one ofthese properties). The proteins that formed the inclusion bodies maythen be renatured by dilution or dialysis with a compatible buffer.Suitable solvents include, but are not limited to, urea (from about 4 Mto about 8 M), formamide (at least about 80%, volume/volume basis), andguanidine hydrochloride (from about 4 M to about 8 M). Some solventsthat are capable of solubilizing aggregate-forming proteins, such as SDS(sodium dodecyl sulfate) and 70% formic acid, are inappropriate for usein this procedure due to the possibility of irreversible denaturation ofthe proteins, accompanied by a lack of immunogenicity and/or activity.Although guanidine hydrochloride and similar agents are denaturants,this denaturation is not irreversible and renaturation may occur uponremoval (by dialysis, for example) or dilution of the denaturant,allowing re-formation of the immunologically and/or biologically activeprotein of interest. After solubilization, the protein can be separatedfrom other bacterial proteins by standard separation techniques.

Alternatively, it is possible to purify proteins from bacteriaperiplasm. Where the protein is exported into the periplasm of thebacteria, the periplasmic fraction of the bacteria can be isolated bycold osmotic shock in addition to other methods known to those of skillin the art (see, Ausubel et al., supra). To isolate recombinant proteinsfrom the periplasm, the bacterial cells are centrifuged to form apellet. The pellet is resuspended in a buffer containing 20% sucrose. Tolyse the cells, the bacteria are centrifuged and the pellet isresuspended in ice-cold 5 mM MgSO₄ and kept in an ice bath forapproximately 10 minutes. The cell suspension is centrifuged and thesupernatant decanted and saved. The recombinant proteins present in thesupernatant can be separated from the host proteins by standardseparation techniques well known to those of skill in the art.

B. Purification of Proteins from Insect Cells

Proteins can also be purified from eukaryotic gene expression systems asdescribed in, e.g., Fernandez and Hoeffler, Gene Expression Systems(1999). In some embodiments, baculovirus expression systems are used toisolate Archipelin proteins or other proteins of the invention.Recombinant cabulaoviruses are generally generated by replacing thepolyhedrin coding sequence of a baculovirus with a gene to be expressed(e.g., an Archipelin polynucleotide). Viruses lacking the polyhedringene have a unique plaque morphology making them easy to recognize. Insome embodiments, a recombinant baculovirus is generated by firstcloning a polynucleotide of interest into a transfer vector (e.g., a pUCbased vector) such that the polynucleotide is operably linked to apolyhedrin promoter. The transfer vector is transfected with wildtypeDNA into an insect cell (e.g., Sf9, Sf21or BT1-TN-5B1-4 cells),resulting in homologous recombination and replacement of the polyhedringene in the wildtype viral DNA with the polynucleotide of interest.Virus can then be generated and plaque purified. Protein expressionresults upon viral infection of insect cells. Expressed proteins can beharvested from cell supernatant if secreted, or from cell lysates ifintracellular. See, e.g., Ausubel et al. and Fernandez and Hoeffler,supra.

C. Standard Protein Separation Techniques For Purifying Proteins

1. Solubility Fractionation

Often as an initial step, and if the protein mixture is complex, aninitial salt fractionation can separate many of the unwanted host cellproteins (or proteins derived from the cell culture media) from therecombinant protein of interest. The salt used for fractionation can be,e.g., ammonium sulfate. Ammonium sulfate precipitates proteins byeffectively reducing the amount of water in the protein mixture.Proteins then precipitate on the basis of their solubility. The morehydrophobic a protein is, the more likely it is to precipitate at lowerammonium sulfate concentrations. A typical protocol is to add saturatedammonium sulfate to a protein solution so that the resultant ammoniumsulfate concentration is between 20-30%. This will precipitate the mosthydrophobic proteins. The precipitate is discarded (unless the proteinof interest is hydrophobic) and ammonium sulfate is added to thesupernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, through either dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

2. Size Differential Filtration

Based on a calculated molecular weight, a protein of greater and lessersize can be isolated using ultrafiltration through membranes ofdifferent pore sizes (for example, Amicon or Millipore membranes). As afirst step, the protein mixture is ultrafiltered through a membrane witha pore size that has a lower molecular weight cut-off than the molecularweight of the protein of interest. The retentate of the ultrafiltrationis then ultrafiltered against a membrane with a molecular cut offgreater than the molecular weight of the protein of interest. Therecombinant protein will pass through the membrane into the filtrate.The filtrate can then be chromatographed as described below.

3. Column Chromatography

The proteins of interest can also be separated from other proteins onthe basis of their size, net surface charge, hydrophobicity and affinityfor ligands. In addition, antibodies raised against proteins can beconjugated to column matrices and the proteins immunopurified. All ofthese methods are well known in the art.

It will be apparent to one of skill that chromatographic techniques canbe performed at any scale and using equipment from many differentmanufacturers (e.g., Pharmacia Biotech).

IV. Detection of Gene Expression

Those of skill in the art will recognize that detection of expression ofArchipelin polynucleotides has many uses. For example, as discussedherein, detection of Archipelin levels in a patient is useful fordiagnosing diabetes or a predisposition for at least some of thepathological effects of diabetes.

A variety of methods of specific DNA and RNA measurement using nucleicacid hybridization techniques are known to those of skill in the art(see, Sambrook, supra). Some methods involve an electrophoreticseparation (e.g., Southern blot for detecting DNA, and Northern blot fordetecting RNA), but measurement of DNA and RNA can also be carried outin the absence of electrophoretic separation (e.g., by dot blot).Southern blot of genomic DNA (e.g., from a human) can be used forscreening for restriction fragment length polymorphism (RFLP) to detectthe presence of a genetic disorder affecting an Archipelin polypeptideof the invention.

The selection of a nucleic acid hybridization format is not critical. Avariety of nucleic acid hybridization formats are known to those skilledin the art. For example, common formats include sandwich assays andcompetition or displacement assays. Hybridization techniques aregenerally described in Hames and Higgins Nucleic Acid Hybridization, APractical Approach, IRL Press (1985); Gall and Pardue, Proc. Natl. AcadSci. U.S.A., 63:378-383 (1969); and John et al. Nature, 223:582-587(1969).

Detection of a hybridization complex may require the binding of a signalgenerating complex to a duplex of target and probe polynucleotides ornucleic acids. Typically, such binding occurs through ligand andanti-ligand interactions as between a ligand-conjugated probe and ananti-ligand conjugated with a signal. The binding of the signalgeneration complex is also readily amenable to accelerations by exposureto ultrasonic energy.

The label may also allow indirect detection of the hybridizationcomplex. For example, where the label is a hapten or antigen, the samplecan be detected by using antibodies. In these systems, a signal isgenerated by attaching fluorescent or enzyme molecules to the antibodiesor in some cases, by attachment to a radioactive label (see, e.g.,Tijssen, “Practice and Theory of Enzyme Immunoassays,” LaboratoryTechniques in Biochemistry and Molecular Biology, Burdon and vanKnippenberg Eds., Elsevier (1985), pp. 9-20).

The probes are typically labeled either directly, as with isotopes,chromophores, lumiphores, chromogens, or indirectly, such as withbiotin, to which a streptavidin complex may later bind. Thus, thedetectable labels used in the assays of the present invention can beprimary labels (where the label comprises an element that is detecteddirectly or that produces a directly detectable element) or secondarylabels (where the detected label binds to a primary label, e.g., as iscommon in immunological labeling). Typically, labeled signal nucleicacids are used to detect hybridization. Complementary nucleic acids orsignal nucleic acids may be labeled by any one of several methodstypically used to detect the presence of hybridized polynucleotides. Themost common method of detection is the use of autoradiography with ³H,¹²⁵I, ³⁵S, ¹⁴C, or ³²P-labeled probes or the like.

Other labels include, e.g., ligands which bind to labeled antibodies,fluorophores, chemiluminescent agents, enzymes, and antibodies which canserve as specific binding pair members for a labeled ligand. Anintroduction to labels, labeling procedures and detection of labels isfound in Polak and Van Noorden Introduction to Immunocytochemistry, 2nded., Springer Verlag, NY (1997); and in Haugland Handbook of FluorescentProbes and Research Chemicals, a combined handbook and cataloguePublished by Molecular Probes, Inc. (1996).

In general, a detector which monitors a particular probe or probecombination is used to detect the detection reagent label. Typicaldetectors include spectrophotometers, phototubes and photodiodes,microscopes, scintillation counters, cameras, film and the like, as wellas combinations thereof. Examples of suitable detectors are widelyavailable from a variety of commercial sources known to persons of skillin the art. Commonly, an optical image of a substrate comprising boundlabeling moieties is digitized for subsequent computer analysis.

Most typically, the amount of, for example, an Archipelin RNA ismeasured by quantitating the amount of label fixed to the solid supportby binding of the detection reagent. Typically, the presence of amodulator during incubation will increase or decrease the amount oflabel fixed to the solid support relative to a control incubation whichdoes not comprise the modulator, or as compared to a baselineestablished for a particular reaction type. Means of detecting andquantitating labels are well known to those of skill in the art.

In some embodiments, the target nucleic acid or the probe is immobilizedon a solid support. Solid supports suitable for use in the assays of theinvention are known to those of skill in the art. As used herein, asolid support is a matrix of material in a substantially fixedarrangement.

A variety of automated solid-phase assay techniques are alsoappropriate. For instance, very large scale immobilized polymer arrays(VLSIPS™), available from Affymetrix, Inc. in Santa Clara, Calif. can beused to detect changes in expression levels of a plurality of genesinvolved in the same regulatory pathways simultaneously. See, Tijssen,supra., Fodor et al. (1991) Science, 251: 767- 777; Sheldon et al.(1993) Clinical Chemistry 39(4): 718-719, and Kozal et al. (1996) NatureMedicine 2(7): 753-759.

Detection can be accomplished, for example, by using a labeled detectionmoiety that binds specifically to duplex nucleic acids (e.g., anantibody that is specific for RNA-DNA duplexes). One example uses anantibody that recognizes DNA-RNA heteroduplexes in which the antibody islinked to an enzyme (typically by recombinant or covalent chemicalbonding). The antibody is detected when the enzyme reacts with itssubstrate, producing a detectable product. Coutlee et al. (1989)Analytical Biochemistry 181:153-162; Bogulavski (1986) etal. J Immunol.Methods 89:123-130; Prooijen-Knegt (1982) Exp. Cell Res. 141:397-407;Rudkin (1976) Nature 265:472-473, Stollar (1970) PNAS 65:993-1000;Ballard (1982) Mol. Immunol. 19:793-799; Pisetsky and Caster (1982) Mol.Immunol. 19:645-650; Viscidi etal. (1988) J Clin. Microbial. 41:199-209;and Kiney et al. (1989) J Clin. Microbiol. 27:6-12 describe antibodiesto RNA duplexes, including homo and heteroduplexes. Kits comprisingantibodies specific for DNA:RNA hybrids are available, e.g., from DigeneDiagnostics, Inc. (Beltsville, Md.).

In addition to available antibodies, one of skill in the art can easilymake antibodies specific for nucleic acid duplexes using existingtechniques, or modify those antibodies which are commercially orpublicly available. In addition to the art referenced above, generalmethods for producing polyclonal and monoclonal antibodies are known tothose of skill in the art (see, e.g., Paul (ed) Fundamental Immunology,Third Edition Raven Press, Ltd., N.Y. (1993); Coligan Current Protocolsin Immunology Wiley/Greene, N.Y. (1991); Harlow and Lane Antibodies. ALaboratory Manual Cold Spring Harbor Press, N.Y. (1989); Stites et al.(eds.) Basic and Clinical Immunology (4th ed.) Lange MedicalPublications, Los Altos, CA, and references cited therein; GodingMonoclonal Antibodies. Principles and Practice (2d ed.) Academic Press,New York, N.Y., (1986); and Kohler and Milstein Nature 256: 495-497(1975)). Other suitable techniques for antibody preparation includeselection of libraries of recombinant antibodies in phage or similarvectors (see, Huse et al. Science 246:1275-1281 (1989); and Ward et al.Nature 341:544-546(1989)). Specific monoclonal and polyclonal antibodiesand antisera will usually bind with a K_(D) of at least about 0.1 μM,preferably at least about 0.01 μM or better, and most typically andpreferably, 0.001 μM or better.

The nucleic acids used in this invention can be either positive ornegative probes. Positive probes bind to their targets and the presenceof duplex formation is evidence of the presence of the target. Negativeprobes fail to bind to the suspect target and the absence of duplexformation is evidence of the presence of the target. For example, theuse of a wild type specific nucleic acid probe or PCR primers may serveas a negative probe in an assay sample where only the nucleotidesequence of interest is present.

The sensitivity of the hybridization assays may be enhanced through useof a nucleic acid amplification system that multiplies the targetnucleic acid being detected. Examples of such systems include thepolymerase chain reaction (PCR) system and the ligase chain reaction(LCR) system. Other methods described in the art are the nucleic acidsequence based amplification (NASBA, Cangene, Mississauga, Ontario) andQ Beta Replicase systems. These systems can be used to directly identifymutants where the PCR or LCR primers are designed to be extended orligated only when a selected sequence is present. Alternatively, theselected sequences can be generally amplified using, for example,nonspecific PCR primers and the amplified target region later probed fora specific sequence indicative of a mutation.

An alternative means for determining the level of expression of thenucleic acids of the present invention is in situ hybridization. In situhybridization assays are well known and are generally described inAngerer et al., Methods Enzymol. 152:649-660 (1987). In an in situhybridization assay, cells, preferentially human pancreatic cells suchas islet cells, are fixed to a solid support, typically a glass slide.If DNA is to be probed, the cells are denatured with heat or alkali. Thecells are then contacted with a hybridization solution at a moderatetemperature to permit annealing of specific probes that are labeled. Theprobes are preferably labeled with radioisotopes or fluorescentreporters.

V. Immunological Detection of Archipelin

In addition to the detection of Archipelin genes and gene expressionusing nucleic acid hybridization technology, one can also useimmunoassays to detect Archipelin polypeptides. Immunoassays can be usedto qualitatively or quantitatively analyze Archipelin. A generaloverview of the applicable technology can be found in Harlow & Lane,Antibodies. A Laboratory Manual (1988).

A. Antibodies to Target Proteins

Methods for producing polyclonal and monoclonal antibodies that reactspecifically with a protein of interest are known to those of skill inthe art (see, e.g., Coligan, supra; and Harlow and Lane, supra; Stiteset al., supra and references cited therein; Goding, supra; and Kohlerand Milstein Nature, 256:495-497 (1975)). Such techniques includeantibody preparation by selection of antibodies from libraries ofrecombinant antibodies in phage or similar vectors (see, Huse et al.,supra; and Ward et al., supra). For example, in order to produceantisera for use in an immunoassay, the protein of interest or anantigenic fragment thereof, is isolated as described herein. Forexample, a recombinant protein is produced in a transformed cell line.An inbred strain of mice or rabbits is immunized with the protein usinga standard adjuvant, such as Freund's adjuvant, and a standardimmunization protocol. Alternatively, a synthetic peptide derived fromthe sequences disclosed herein and conjugated to a carrier protein canbe used as an immunogen.

Polyclonal sera are collected and titered against the immunogen proteinin an immunoassay, for example, a solid phase immunoassay with theimmunogen immobilized on a solid support. Polyclonal antisera with atiter of 10⁴ or greater are selected and tested for theircrossreactivity against non-Archipelin proteins or even other homologousproteins from other organisms, using a competitive binding immunoassay.Specific monoclonal and polyclonal antibodies and antisera will usuallybind with a KD of at least about 0.1 μM, more usually at least about 1μM, preferably at least about 0.1 μM or better, and most preferably,0.01 μM or better.

For preparation of antibodies, e.g., recombinant, monoclonal, orpolyclonal antibodies, many technique known in the art can be used (see,e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al.,Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan,Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, ALaboratory Manual (1988); and Goding, Monoclonal Antibodies. Principlesand Practice (2d ed. 1986)). The genes encoding the heavy and lightchains of an antibody of interest can be cloned from a cell, e.g., thegenes encoding a monoclonal antibody can be cloned from a hybridoma andused to produce a recombinant monoclonal antibody. Gene librariesencoding heavy and light chains of monoclonal antibodies can also bemade from hybridoma or plasma cells. Random combinations of the heavyand light chain gene products generate a large pool of antibodies withdifferent antigenic specificity (see, e.g., Kuby, Immunology (3rd ed.1997)). Techniques for the production of single chain antibodies orrecombinant antibodies (U.S. Pat. No. 4,946,778, U.S. Pat. No.4,816,567) can be adapted to produce antibodies to polypeptides of thisinvention. Also, transgenic mice, or other organisms such as othermammals, may be used to express humanized or human antibodies (see,e.g., U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;5,633,425; 5,661,016, Marks et al., Bio/Technology 10:779-783 (1992);Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13(1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996);Neuberger, Nature Biotechnology 14:826 (1996); and Lonberg & Huszar,Intern. Rev. Immunol. 13:65-93 (1995)). Alternatively, phage displaytechnology can be used to identify antibodies and heteromeric Fabfragments that specifically bind to selected antigens (see, e.g.,McCafferty et al., Nature 348:552-554 (1990); Marks et al, Biotechnology10:779-783 (1992)). Antibodies can also be made bispecific, i.e., ableto recognize two different antigens (see, e.g., WO 93/08829, Trauneckeret al., EMBO J. 10:3655-3659 (1991); and Suresh et al., Methods inEnzymology 121:210 (1986)). Antibodies can also be heteroconjugates,e.g., two covalently joined antibodies, or immunotoxins (see, e.g., U.S.Pat. No. 4,676,980, WO 91/00360; WO 92/200373; and EP 03089).

Methods for humanizing or primatizing non-human antibodies are wellknown in the art. Generally, a humanized antibody has one or more aminoacid residues introduced into it from a source which is non-human. Thesenon-human amino acid residues are often referred to as import residues,which are typically taken from an import variable domain. Humanizationcan be essentially performed following the method of Winter andco-workers (see, e.g., Jones et al., Nature 321:522-525 (1986);Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science239:1534-1536 (1988) and Presta, Curr. Op. Struct. Biol. 2:593-596(1992)), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody. Accordingly, such humanizedantibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies.

A number of proteins of the invention comprising immunogens may be usedto produce antibodies specifically or selectively reactive with theproteins of interest. Recombinant protein is an exemplary immunogen forthe production of monoclonal or polyclonal antibodies. Naturallyoccurring protein may also be used either in pure or impure form.Synthetic peptides made using the protein sequences described herein mayalso be used as an immunogen for the production of antibodies to theprotein. Recombinant protein can be expressed in eukaryotic orprokaryotic cells and purified as generally described supra. The productis then injected into an animal capable of producing antibodies. Eithermonoclonal or polyclonal antibodies may be generated for subsequent usein immunoassays to measure the protein.

Methods of production of polyclonal antibodies are known to those ofskill in the art. In brief, an immunogen, preferably a purified protein,is mixed with an adjuvant and animals are immunized. The animal's immuneresponse to the immunogen preparation is monitored by taking test bleedsand determining the titer of reactivity to the Archipelin of interest.When appropriately high titers of antibody to the immunogen areobtained, blood is collected from the animal and antisera are prepared.Further fractionation of the antisera to enrich for antibodies reactiveto the protein can be done if desired (see, Harlow and Lane, supra).

Monoclonal antibodies may be obtained using various techniques familiarto those of skill in the art. Typically, spleen cells from an animalimmunized with a desired antigen are immortalized, commonly by fusionwith a myeloma cell (see, Kohler and Milstein, Eur. J Immunol. 6:511-519(1976)): Alternative methods of immortalization include, e.g.,transformation with Epstein Barr Virus, oncogenes, or retroviruses, orother methods well known in the art. Colonies arising from singleimmortalized cells are screened for production of antibodies of thedesired specificity and affinity for the antigen, and yield of themonoclonal antibodies produced by such cells may be enhanced by varioustechniques, including injection into the peritoneal cavity of avertebrate host. Alternatively, one may isolate DNA sequences whichencode a monoclonal antibody or a binding fragment thereof by screeninga DNA library from human B cells according to the general protocoloutlined by Huse et al., supra.

Once target protein specific antibodies are available, the protein canbe measured by a variety of immunoassay methods with qualitative andquantitative results available to the clinician. For a review ofimmunological and immunoassay procedures in general see, Stites, supra.Moreover, the immunoassays of the present invention can be performed inany of several configurations, which are reviewed extensively in MaggioEnzyme Immunoassay, CRC Press, Boca Raton, Fla. (1980); Tijssen, supra;and Harlow and Lane, supra.

Immunoassays to measure target proteins in a human sample may use apolyclonal antiserum which was raised to the protein (e.g., SEQ ID NO:2,SEQ ID NO:7 and SEQ ID NOs:9-14) at least partially encoded by asequence described herein (e.g., SEQ ID NO: 1 and SEQ ID NO:8) or afragment thereof. This antiserum is selected to have lowcross-reactivity against non-Archipelin proteins and any suchcross-reactivity is removed by immunoabsorption prior to use in theimmunoassay.

Polyclonal antibodies that specifically bind to an Archipelin ofinterest from a particular species can be made by subtracting outcross-reactive antibodies using Archipelin homologs. In an analogousfashion, antibodies specific to a particular Archipelin (e.g., the humanArchipelin polypeptide) can be obtained in an organism with multipleArchipelin genes by subtracting out cross-reactive antibodies usingother Archipelin.

B. Immunological Binding Assays

In some embodiments, a protein of interest is detected and/or quantifiedusing any of a number of well known immunological binding assays (see,e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168).For a review of the general immunoassays, see also Asai Methods in CellBiology Volume 37.Antibodies in Cell Biology, Academic Press, Inc. N.Y.(1993); Stites, supra. Immunological binding assays (or immunoassays)typically utilize a “capture agent” to specifically bind to and oftenimmobilize the analyte (in this case an Archipelin of the presentinvention or antigenic subsequences thereof). The capture agent is amoiety that specifically binds to the analyte. In some embodiments, thecapture agent is an antibody that specifically binds, for example, anArchipelin polypeptide of the invention. The antibody (e.g.,anti-Archipelin antibody) may be produced by any of a number of meanswell known to those of skill in the art and as described above.

Immunoassays also often utilize a labeling agent to specifically bind toand label the binding complex formed by the capture agent and theanalyte. The labeling agent may itself be one of the moieties comprisingthe antibody/analyte complex. Thus, the labeling agent may be a labeledArchipelin polypeptide or a labeled anti-Archipelin receptor antibody.Alternatively, the labeling agent may be a third moiety, such as anotherantibody, that specifically binds to the antibody/protein complex.

In some embodiments, the labeling agent is a second antibody bearing alabel. Alternatively, the second antibody may lack a label, but it may,in turn, be bound by a labeled third antibody specific to antibodies ofthe species from which the second antibody is derived. The secondantibody can be modified with a detectable moiety, such as biotin, towhich a third labeled molecule can specifically bind, such asenzyme-labeled streptavidin.

Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G, can also be used as the labelagents. These proteins are normal constituents of the cell walls ofstreptococcal bacteria. They exhibit a strong non-immunogenic reactivitywith immunoglobulin constant regions from a variety of species (see,generally, Kronval, et al. J Immunol., 111: 1401-1406 (1973); andAkerstrom, et al. J. Immunol., 135:2589-2542 (1985)).

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. The incubation time will depend upon the assay format, analyte,volume of solution, concentrations, and the like. Usually, the assayswill be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 10° C. to 40° C.

1. Non-Competitive Assay Formats

Immunoassays for detecting proteins of interest from tissue samples maybe either competitive or noncompetitive. Noncompetitive immunoassays areassays in which the amount of captured analyte (in this case theprotein) is directly measured. In one “sandwich” assay, for example, thecapture agent (e.g., anti-Archipelin antibodies) can be bound directlyto a solid substrate where it is immobilized. These immobilizedantibodies then capture the Archipelin present in the test sample. TheArchipelin thus immobilized is then bound by a labeling agent, such as asecond anti-Archipelin receptor antibody bearing a label. Alternatively,the second antibody may lack a label, but it may, in turn, be bound by alabeled third antibody specific to antibodies of the species from whichthe second antibody is derived. The second can be modified with adetectable moiety, such as biotin, to which a third labeled molecule canspecifically bind, such as enzyme-labeled streptavidin.

2. Competitive Assay Formats

In competitive assays, the amount of target protein (analyte) present inthe sample is measured indirectly by measuring the amount of an added(exogenous) analyte (i.e., the Archipelin of interest) displaced (orcompeted away) from a capture agent (i.e., anti antibody) by the analytepresent in the sample. In one competitive assay, a known amount of, inthis case, the protein of interest is added to the sample and the sampleis then contacted with a capture agent, in this case an antibody thatspecifically binds to the Archipelin of interest. The amount ofArchipelin bound to the antibody is inversely proportional to theconcentration of Archipelin present in the sample. In some embodiments,the antibody is immobilized on a solid substrate. The amount of theArchipelin bound to the antibody may be determined either by measuringthe amount of subject protein present in a Archipelin protein/antibodycomplex or, alternatively, by measuring the amount of remaininguncomplexed protein. The amount of Archipelin protein may be detected byproviding a labeled Archipelin protein molecule.

A hapten inhibition assay is another exemplary competitive assay. Inthis assay, a known analyte, in this case the target protein, isimmobilized on a solid substrate. A known amount of anti-Archipelinantibody is added to the sample, and the sample is then contacted withthe immobilized target. In this case, the amount of anti-Archipelinantibody bound to the immobilized Archipelin is inversely proportionalto the amount of Archipelin protein present in the sample. Again, theamount of immobilized antibody may be detected by detecting either theimmobilized fraction of antibody or the fraction of the antibody thatremains in solution. Detection may be direct where the antibody islabeled or indirect by the subsequent addition of a labeled moiety thatspecifically binds to the antibody as described above.

Immunoassays in the competitive binding format can be used forcross-reactivity determinations. For example, the protein encoded by thesequences described herein can be immobilized on a solid support.Proteins are added to the assay which compete with the binding of theantisera to the immobilized antigen. The ability of the above proteinsto compete with the binding of the antisera to the immobilized proteinis compared to that of the protein encoded by any of the sequencesdescribed herein. The percent cross-reactivity for the above proteins iscalculated, using standard calculations. Those antisera with less than10% cross-reactivity with each of the proteins listed above are selectedand pooled. The cross-reacting antibodies are optionally removed fromthe pooled antisera by immunoabsorption with the considered proteins,e.g., distantly related homologs.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein,thought to be perhaps a protein of the present invention, to theimmunogen protein. In order to make this comparison, the two proteinsare each assayed at a wide range of concentrations and the amount ofeach protein required to inhibit 50% of the binding of the antisera tothe immobilized protein is determined. If the amount of the secondprotein required is less than 10 times the amount of the proteinpartially encoded by a sequence herein that is required, then the secondprotein is said to specifically bind to an antibody generated to animmunogen consisting of the target protein.

3. Other Assay Formats

In some embodiments, western blot (immunoblot) analysis is used todetect and quantify the presence of an Archipelin of the invention inthe sample. The technique generally comprises separating sample proteinsby gel electrophoresis on the basis of molecular weight, transferringthe separated proteins to a suitable solid support (such as, e.g., anitrocellulose filter, a nylon filter, or a derivatized nylon filter)and incubating the sample with the antibodies that specifically bind theprotein of interest. For example, the anti-Archipelin antibodiesspecifically bind to the Archipelin on the solid support. Theseantibodies may be directly labeled or alternatively may be subsequentlydetected using labeled antibodies (e.g., labeled sheep anti-mouseantibodies) that specifically bind to the antibodies against the proteinof interest.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see, Monroe et al.(1986) Amer. Clin. Prod. Rev. 5:34-41).

4. Labels

The particular label or detectable group used in the assay is not acritical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most labels useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., Dynabeads™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or32P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and calorimetric labels such ascolloidal gold or colored glass or plastic (e.g., polystyrene,polypropylene, latex, etc.) beads.

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on the sensitivity required, the ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Themolecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorescent compound.A variety of enzymes and fluorescent compounds can be used with themethods of the present invention and are well-known to those of skill inthe art (for a review of various labeling or signal producing systemswhich may be used, see, e.g., U.S. Pat. No. 4,391,904).

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected directly by observing the color associated withthe label. Thus, in various dipstick assays, conjugated gold oftenappears pink, while various conjugated beads appear the color of thebead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need to be labeled and the presence ofthe target antibody is detected by simple visual inspection.

VI. Screening for Modulators of Archipelin

Modulators of Archipelin, i.e. agonists or antagonists or agents ofArchipelin activity or modulators of Archipelin polypeptide orpolynucleotide expression, are useful for treating a number of humandiseases, including diabetes. Administration of Archipelin agonists oragents that increase expression of Archipelin can be used to treatdiabetic patients. For example, insufficient Archipelin due tofunctional impairment of islets may contribute to some of thepathologies associated with diabetes. Thus, restoration of Archipelinameliorates some of these pathologies.

Conversely, under conditions of islet hyperactivity, such as occurs inan insulin resistant states, islet expansion may lead to overproductionof Archipelin. Overproduction leads to a different set of deleteriousphysiological effects that can be relieved by Archipelin antagonists.Archipelin agonists or antagonists may have beneficial physiologicaleffects in diabetes whether or not the endogenous level of the peptideis abnormal.

A. Methods for identifying Modulators of Archipelin

A number of different screening protocols can be utilized to identifyagents that modulate the level of expression or activity of Archipelinin cells, particularly mammalian cells, and especially human cells. Ingeneral terms, the screening methods involve screening a plurality ofagents to identify an agent that modulates the activity of Archipelin bybinding to Archipelin, preventing an inhibitor from binding toArchipelin or activating expression of Archipelin, for example.

1. Archipelin Binding Assays

Preliminary screens can be conducted by screening for compounds capableof binding to Archipelin, as at least some of the compounds soidentified are likely Archipelin activators. The binding assays usuallyinvolve contacting an Archipelin protein with one or more test compoundsand allowing sufficient time for the protein and test compounds to forma binding complex. Any binding complexes formed can be detected usingany of a number of established analytical techniques. Protein bindingassays include, but are not limited to, methods that measureco-precipitation, co-migration on non-denaturing SDS-polyacrylamidegels, and co-migration on Western blots (see, e.g., Bennet, J.P. andYamamura, H.I. (1985) “Neurotransmitter, Hormone or Drug ReceptorBinding Methods,” in Neurotransmitter Receptor Binding (Yamamura, H.I.,et al., eds.), pp. 61-89) as well as phage display and other bindingassays known to those of skill in the art. The Archipelin proteinutilized in such assays can be naturally expressed, cloned orsynthesized Archipelin. In some embodiments, two hybrid assays, or otherexpression-based in vivo binding assays can be used. See, e.g., Fields,et al., Nature 340(6230):245-6 (1989).

Binding assays are also useful, e.g., for identifying endogenousproteins that interact with Archipelin. For example, receptors that bindArchipelin can be identified in binding assays.

2. Expression Assays

Certain screening methods involve screening for a compound thatup-regulates the expression of Archipelin. Such methods generallyinvolve conducting cell-based assays in which test compounds arecontacted with one or more cells expressing Archipelin and thendetecting an increase or decrease in Archipelin expression (eithertranscript or translation product). Some assays are performed withpancreatic islet cells, or other cells, that express endogenousArchipelin.

Archipelin expression can be detected in a number of different ways. Asdescribed herein, the expression level of Archipelin in a cell can bedetermined by probing the mRNA expressed in a cell with a probe thatspecifically hybridizes with a transcript (or complementary nucleic acidderived therefrom) of Archipelin. Probing can be conducted by lysing thecells and conducting Northern blots or without lysing the cells using insitu-hybridization techniques (see above). Alternatively, Archipelinprotein can be detected using immunological methods in which a celllysate is probe with antibodies that specifically bind to Archipelin.

Other cell-based assays are reporter assays conducted with cells that donot express Archipelin. Certain of these assays are conducted with aheterologous nucleic acid construct that includes a Archipelin promoterthat is operably linked to a reporter gene that encodes a detectableproduct. A number of different reporter genes can be utilized. Somereporters are inherently detectable. An example of such a reporter isgreen fluorescent protein that emits fluorescence that can be detectedwith a fluorescence detector. Other reporters generate a detectableproduct. Often such reporters are enzymes. Exemplary enzyme reportersinclude, but are not limited to, β-glucuronidase, CAT (chloramphenicolacetyl transferase; Alton and Vapnek (1979) Nature 282:864-869),luciferase, β-galactosidase and alkaline phosphatase (Toh, et al. (1980)Eur. J Biochem. 182:231-238; and Hall et al. (1983) J Mol. Appl. Gen.2:101).

In these assays, cells harboring the reporter construct are contactedwith a test compound. A test compound that either modulates the activityof the promoter by binding to it or triggers a cascade that produces amolecule that modulates the promoter causes expression of the detectablereporter. Certain other reporter assays are conducted with cells thatharbor a heterologous construct that includes a transcriptional controlelement that activates expression of Archipelin and a reporter operablylinked thereto. Here, too, an agent that binds to the transcriptionalcontrol element to activate expression of the reporter or that triggersthe formation of an agent that binds to the transcriptional controlelement to activate reporter expression, can be identified by thegeneration of signal associated with reporter expression.

The level of expression or activity can be compared to a baseline value.As indicated above, the baseline value can be a value for a controlsample or a statistical value that is representative of Archipelinexpression levels for a control population (e.g., healthy individualsnot having or at risk for type 1 or type 2 diabetes). Expression levelscan also be determined for cells that do not express Archipelin as anegative control. Such cells generally are otherwise substantiallygenetically the same as the test cells.

A variety of different types of cells can be utilized in the reporterassays. As stated above, certain cells are nerve cells that express anendogenous Archipelin. Cells not expressing Archipelin can beprokaryotic, but preferably are eukaryotic. The eukaryotic cells can beany of the cells typically utilized in generating cells that harborrecombinant nucleic acid constructs. Exemplary eukaryotic cells include,but are not limited to, yeast, and various higher eukaryotic cells suchas the COS, CHO and HeLa cell lines.

Various controls can be conducted to ensure that an observed activity isauthentic including running parallel reactions with cells that lack thereporter construct or by not contacting a cell harboring the reporterconstruct with test compound. Compounds can also be further validated asdescribed below.

3. Validation

Compounds that are initially identified by any of the foregoingscreening methods can be further tested to validate the apparentactivity. Preferably such studies are conducted with suitable animalmodels. The basic format of such methods involves administering a leadcompound identified during an initial screen to an animal that serves asa model for humans and then determining if Archipelin is in factmodulated. The animal models utilized in validation studies generallyare mammals of any kind. Specific examples of suitable animals include,but are not limited to, primates, mice and rats.

B. Modulators of Archipelin

The compounds tested as modulators of Archipelin can be any smallchemical compound, or a biological entity, such as a protein, sugar,nucleic acid or lipid. Alternatively, modulators can be geneticallyaltered versions of an Archipelin gene or gene product. Typically, testcompounds will be small chemical molecules and peptides. Essentially anychemical compound can be used as a potential modulator or ligand in theassays of the invention, although most often compounds that can bedissolved in aqueous or organic (especially DMSO-based) solutions areused. The assays are designed to screen large chemical libraries byautomating the assay steps and providing compounds from any convenientsource to assays, which are typically run in parallel (e.g., inmicrotiter formats on microtiter plates in robotic assays). It will beappreciated that there are many suppliers of chemical compounds,including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.),Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika(Buchs, Switzerland) and the like.

In some embodiments, high throughput screening methods involve providinga combinatorial chemical or peptide library containing a large number ofpotential therapeutic compounds (potential modulator or ligandcompounds). Such “combinatorial chemical libraries” or “ligandlibraries” are then screened in one or more assays, as described herein,to identify those library members (particular chemical species orsubclasses) that display a desired characteristic activity. Thecompounds thus identified can serve as conventional “lead compounds” orcan themselves be used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091),benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat.Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagiharaet al., J Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimeticswith glucose scaffolding (Hirschmann et al., J Amer. Chem. Soc.114:9217-9218 (1992)), analogous organic syntheses of small compoundlibraries (Chen et al., J Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J Org. Chem. 59:658 (1994)), nucleic acidlibraries (see Ausubel, Berger and Sambrook, all supra), peptide nucleicacid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibody libraries(see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314 (1996)and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al.,Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), smallorganic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, 5,288,514, and thelike).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g, 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Tripos, Inc., St. Louis, Mo., 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

C. Solid Phase and Soluble High Throughput Assays

In the high throughput assays of the invention, it is possible to screenup to several thousand different modulators or ligands in a single day.In particular, each well of a microtiter plate can be used to run aseparate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 100 (e.g., 96) modulators. If 1536 well plates areused, then a single plate can easily assay from about 100 to about 1500different compounds. It is possible to assay several different platesper day; assay screens for up to about 6,000-20,000 different compoundsare possible using the integrated systems of the invention. Morerecently, microfluidic approaches to reagent manipulation have beendeveloped.

The molecule of interest can be bound to the solid state component,directly or indirectly, via covalent or non covalent linkage, e.g., viaa tag. The tag can be any of a variety of components. In general, amolecule that binds the tag (a tag binder) is fixed to a solid support,and the tagged molecule of interest (e.g., Archipelin) is attached tothe solid support by interaction of the tag and the tag binder.

A number of tags and tag binders can be used, based upon known molecularinteractions well described in the literature. For example, where a taghas a natural binder, for example, biotin, protein A, or protein G, itcan be used in conjunction with appropriate tag binders (avidin,streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.)Antibodies to molecules with natural binders such as biotin are alsowidely available and appropriate tag binders (see, SIGMA Immunochemicals1998 catalogue SIGMA, St. Louis Mo.).

Similarly, any haptenic or antigenic compound can be used in combinationwith an appropriate antibody to form a tag/tag binder pair. Thousands ofspecific antibodies are commercially available and many additionalantibodies are described in the literature. For example, in one commonconfiguration, the tag is a first antibody and the tag binder is asecond antibody which recognizes the first antibody. In addition toantibody-antigen interactions, receptor-ligand interactions are alsoappropriate as tag and tag-binder pairs, such as agonists andantagonists of cell membrane receptors (e.g., cell receptor-ligandinteractions such as transferrin, c-kit, viral receptor ligands,cytokine receptors, chemokine receptors, interleukin receptors,immunoglobulin receptors and antibodies, the cadherin family, theintegrin family, the selectin family, and the like; see, e.g., Pigott &Power, The Adhesion Molecule Facts Book I (1993)). Similarly, toxins andvenoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),intracellular receptors (e.g., which mediate the effects of varioussmall ligands, including steroids, thyroid hormone, retinoids andvitamin D; peptides), drugs, lectins, sugars, nucleic acids (both linearand cyclic polymer configurations), oligosaccharides, proteins,phospholipids and antibodies can all interact with various cellreceptors.

Synthetic polymers, such as polyurethanes, polyesters, polycarbonates,polyureas, polyamides, polyethyleneimines, polyarylene sulfides,polysiloxanes, polyimides, and polyacetates can also form an appropriatetag or tag binder. Many other tag/tag binder pairs are also useful inassay systems described herein, as would be apparent to one of skillupon review of this disclosure.

Common linkers such as peptides, polyethers, and the like can also serveas tags, and include polypeptide sequences, such as poly Gly sequencesof between about 5 and 200 amino acids (SEQ ID NO:53). Such flexiblelinkers are known to those of skill in the art. For example,poly(ethylene glycol) linkers are available from Shearwater Polymers,Inc., Huntsville, Ala. These linkers optionally have amide linkages,sulfhydryl linkages, or heterofunctional linkages.

Tag binders are fixed to solid substrates using any of a variety ofmethods currently available. Solid substrates are commonly derivatizedor functionalized by exposing all or a portion of the substrate to achemical reagent which fixes a chemical group to the surface which isreactive with a portion of the tag binder. For example, groups which aresuitable for attachment to a longer chain portion would include amines,hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes andhydroxyalkylsilanes can be used to functionalize a variety of surfaces,such as glass surfaces. The construction of such solid phase biopolymerarrays is well described in the literature (see, e.g., Merrifield, J Am.Chem. Soc. 85:2149-2154 (1963) (describing solid phase synthesis of,e.g., peptides); Geysen et al., J Immun. Meth. 102:259-274 (1987)(describing synthesis of solid phase components on pins); Frank andDoring, Tetrahedron 44:60316040 (1988) (describing synthesis of variouspeptide sequences on cellulose disks); Fodor et al., Science,251:767-777 (1991); Sheldon et al., Clinical Chemistry 39(4):718-719(1993); and Kozal etal., Nature Medicine 2(7):753759 (1996) (alldescribing arrays of biopolymers fixed to solid substrates).Non-chemical approaches for fixing tag binders to substrates includeother common methods, such as heat, cross-linking by UV radiation, andthe like.

The invention provides in vitro assays for identifying, in a highthroughput format, compounds that can modulate the expression oractivity of Archipelin. Control reactions that measure Archipelinactivity of the cell in a reaction that does not include a potentialmodulator are optional, as the assays are highly uniform. Such optionalcontrol reactions are appropriate and increase the reliability of theassay. Accordingly, in some embodiments, the methods of the inventioninclude such a control reaction. For each of the assay formatsdescribed, “no modulator” control reactions which do not include amodulator provide a background level of binding activity.

In some assays it will be desirable to have positive controls to ensurethat the components of the assays are working properly. At least twotypes of positive controls are appropriate. First, a known activator ofArchipelin of the invention can be incubated with one sample of theassay, and the resulting increase in signal resulting from an increasedexpression level or activity of Archipelin determined according to themethods herein. Second, a known inhibitor of Archipelin can be added,and the resulting decrease in signal for the expression or activity ofArchipelin can be similarly detected. It will be appreciated thatmodulators can also be combined with activators or inhibitors to findmodulators which inhibit the increase or decrease that is otherwisecaused by the presence of the known modulator of Archipelin.

D. Computer-Based Assays

Yet another assay for compounds that modulate the activity of Archipelininvolves computer assisted drug design, in which a computer system isused to generate a three-dimensional structure of Archipelin based onthe structural information encoded by its amino acid sequence. The inputamino acid sequence interacts directly and actively with apre-established algorithm in a computer program to yield secondary,tertiary, and quaternary structural models of the protein. Similaranalyses can be performed on potential receptors of Archipelin. Themodels of the protein structure are then examined to identify regions ofthe structure that have the ability to bind, e.g., Archipelin. Theseregions are then used to identify polypeptides that bind to Archipelin.

The three-dimensional structural model of the protein is generated byentering protein amino acid sequences of at least 10 amino acid residuesor corresponding nucleic acid sequences encoding a potential Archipelinreceptor into the computer system. The amino acid sequences encoded bythe nucleic acid sequences provided herein represent the primarysequences or subsequences of the proteins, which encode the structuralinformation of the proteins. At least 10 residues of an amino acidsequence (or a nucleotide sequence encoding 10 amino acids) are enteredinto the computer system from computer keyboards, computer readablesubstrates that include, but are not limited to, electronic storagemedia (e.g., magnetic diskettes, tapes, cartridges, and chips), opticalmedia (e.g., CD ROM), information distributed by internet sites, and byRAM. The three-dimensional structural model of the protein is thengenerated by the interaction of the amino acid sequence and the computersystem, using software known to those of skill in the art.

The amino acid sequence represents a primary structure that encodes theinformation necessary to form the secondary, tertiary and quaternarystructure of the protein of interest. The software looks at certainparameters encoded by the primary sequence to generate the structuralmodel. These parameters are referred to as “energy terms,” and primarilyinclude electrostatic potentials, hydrophobic potentials, solventaccessible surfaces, and hydrogen bonding. Secondary energy termsinclude van der Waals potentials. Biological molecules form thestructures that minimize the energy terms in a cumulative fashion. Thecomputer program is therefore using these terms encoded by the primarystructure or amino acid sequence to create the secondary structuralmodel.

The tertiary structure of the protein encoded by the secondary structureis then formed on the basis of the energy terms of the secondarystructure. The user at this point can enter additional variables such aswhether the protein is membrane bound or soluble, its location in thebody, and its cellular location, e.g., cytoplasmic, surface, or nuclear.These variables along with the energy terms of the secondary structureare used to form the model of the tertiary structure. In modeling thetertiary structure, the computer program matches hydrophobic faces ofsecondary structure with like, and hydrophilic faces of secondarystructure with like.

Once the structure has been generated, potential ligand binding regionsare identified by the computer system. Three-dimensional structures forpotential ligands are generated by entering amino acid or nucleotidesequences or chemical formulas of compounds, as described above. Thethree-dimensional structure of the potential ligand is then compared tothat of Archipelin to identify binding sites of Archipelin. Bindingaffinity between the protein and ligands is determined using energyterms to determine which ligands have an enhanced probability of bindingto the protein.

Computer systems are also used to screen for mutations, polymorphicvariants, alleles and interspecies homologs of genes encoding anArchipelin polypeptide of the invention. Such mutations can beassociated with disease states or genetic traits. As described above,GeneChip™ and related technology can also be used to screen formutations, polymorphic variants, alleles and interspecies homologs. Oncethe variants are identified, diagnostic assays can be used to identifypatients having such mutated genes. Identification of the mutatedArchipelin genes involves receiving input of a first amino acid sequenceof a Archipelin (or of a first nucleic acid sequence encoding aArchipelin of the invention), e.g., any amino acid sequence having atleast 60%, optionally at least 85%, identity with the amino acidsequence of the polypeptide encoded by the nucleic acid sequence setforth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:8, orconservatively modified versions thereof. The sequence is entered intothe computer system as described above. The first nucleic acid or aminoacid sequence is then compared to a second nucleic acid or amino acidsequence that has substantial identity to the first sequence. The secondsequence is entered into the computer system in the manner describedabove. Once the first and second sequences are compared, nucleotide oramino acid differences between the sequences are identified. Suchsequences can represent allelic differences in various Archipelin genes,and mutations associated with disease states and genetic traits.

VII. Composistions, Kits and Integrated Systems

The invention provides compositions, kits and integrated systems forpracticing the assays described herein using nucleic acids encoding theArchipelin polypeptides of the invention, or Archipelin proteins,anti-Archipelin antibodies, etc.

The invention provides assay compositions for use in solid phase assays;such compositions can include, for example, one or more nucleic acidsencoding an Archipelin immobilized on a solid support, and a labelingreagent. In each case, the assay compositions can also includeadditional reagents that are desirable for hybridization. Modulators ofexpression or activity of an Archipelin of the invention can also beincluded in the assay compositions. Solid supports include, e.g., petriplates, microtiter dishes and microarrays.

The invention also provides kits for carrying out the assays of theinvention. The kits typically include a probe which comprises anantibody that specifically binds to Archipelin or a polynucleotidesequence encoding an Archipelin polypeptide, and a label for detectingthe presence of the probe. The kits may include several polynucleotidesequences encoding Archipelin polypeptides of the invention. Kits caninclude any of the compositions noted above, and optionally furtherinclude additional components such as instructions to practice ahigh-throughput method of assaying for an effect on expression of thegenes encoding the Archipelin polypeptides of the invention, or onactivity of the Archipelin polypeptides of the invention, one or morecontainers or compartments (e.g., to hold the probe, labels, or thelike), a control modulator of the expression or activity of Archipelinpolypeptides, a robotic armature for mixing kit components or the like.

The invention also provides integrated systems for high-throughputscreening of potential modulators for an effect on the expression oractivity of the Archipelin polypeptides of the invention. The systemstypically include a robotic armature which transfers fluid from a sourceto a destination, a controller which controls the robotic armature, alabel detector, a data storage unit which records label detection, andan assay component such as a microtiter dish comprising a well having areaction mixture or a substrate comprising a fixed nucleic acid orimmobilization moiety.

A number of robotic fluid transfer systems are available, or can easilybe made from existing components. For example, a Zymate XP (ZymarkCorporation; Hopkinton, Mass.) automated robot using a Microlab 2200(Hamilton; Reno, Nev.) pipetting station can be used to transferparallel samples to 96 well microtiter plates to set up several parallelsimultaneous binding assays.

Optical images viewed (and, optionally, recorded) by a camera or otherrecording device (e.g., a photodiode and data storage device) areoptionally further processed in any of the embodiments herein, e.g., bydigitizing the image and storing and analyzing the image on a computer.A variety of commercially available peripheral equipment and software isavailable for digitizing, storing and analyzing a digitized video ordigitized optical image, e.g., using PC (Intel x86 or Pentiumchip-compatible DOS®, OS2® WINDOWS®, WINDOWS NT® or WINDOWS95® basedcomputers), MACINTOSH®, or UNIX® based (e.g., SUN® work station)computers.

One conventional system carries light from the specimen field to acooled charge-coupled device (CCD) camera, in common use in the art. ACCD camera includes an array of picture elements (pixels). The lightfrom the specimen is imaged on the CCD. Particular pixels correspondingto regions of the specimen (e.g., individual hybridization sites on anarray of biological polymers) are sampled to obtain light intensityreadings for each position. Multiple pixels are processed in parallel toincrease speed. The apparatus and methods of the invention are easilyused for viewing any sample, e.g., by fluorescent or dark fieldmicroscopic techniques.

VIII. Gene Therapy Applications

A variety of human diseases can be treated by therapeutic approachesthat involve stably introducing a gene into a human cell such that thegene is transcribed and the gene product is produced in the cell.Diseases amenable to treatment by this approach include inheriteddiseases, including those in which the defect is in a single gene. Genetherapy is also useful for treatment of acquired diseases and otherconditions. For discussions on the application of gene therapy towardsthe treatment of genetic as well as acquired diseases, see, MillerNature 357:455-460 (1992); and Mulligan Science 260:926-932 (1993).

In the context of the present invention, gene therapy can be used fortreating a variety of disorders and/or diseases in which Archipelin hasbeen implicated. For example, introduction by gene therapy ofpolynucleotides encoding an Archipelin polypeptide of the invention canbe used to treat, e.g., diabetes.

A. Vectors for Gene Delivery

For delivery to a cell or organism, the nucleic acids of the inventioncan be incorporated into a vector. Examples of vectors used for suchpurposes include expression plasmids capable of directing the expressionof the nucleic acids in the target cell. In other instances, the vectoris a viral vector system wherein the nucleic acids are incorporated intoa viral genome that is capable of transfecting the target cell. Inembodiments, the nucleic acids can be operably linked to expression andcontrol sequences that can direct expression of the gene in the desiredtarget host cells. Thus, one can achieve expression of the nucleic acidunder appropriate conditions in the target cell.

B. Gene Delivery Systems

Viral vector systems useful in the expression of the nucleic acidsinclude, for example, naturally occurring or recombinant viral vectorsystems. Depending upon the particular application, suitable viralvectors include replication competent, replication deficient, andconditionally replicating viral vectors. For example, viral vectors canbe derived from the genome of human or bovine adenoviruses, vacciniavirus, herpes virus, adeno-associated virus, minute virus of mice (MVM),HIV, sindbis virus, and retroviruses (including but not limited to Roussarcoma virus), and MoMLV. Typically, the genes of interest are insertedinto such vectors to allow packaging of the gene construct, typicallywith accompanying viral DNA, followed by infection of a sensitive hostcell and expression of the gene of interest.

As used herein, “gene delivery system” refers to any means for thedelivery of a nucleic acid of the invention to a target cell. In someembodiments of the invention, nucleic acids are conjugated to a cellreceptor ligand for facilitated uptake (e.g., invagination of coatedpits and internalization of the endosome) through an appropriate linkingmoiety, such as a DNA linking moiety (Wu et al., J. Biol. Chem.263:14621 -14624 (1988); WO 92/06180). For example, nucleic acids can belinked through a polylysine moiety to asialo-oromucocid, which is aligand for the asialoglycoprotein receptor of hepatocytes.

Similarly, viral envelopes used for packaging gene constructs thatinclude the nucleic acids of the invention can be modified by theaddition of receptor ligands or antibodies specific for a receptor topermit receptor-mediated endocytosis into specific cells (see, e.g., WO93/20221, WO 93/14188, and WO 94/06923). In some embodiments of theinvention, the DNA constructs of the invention are linked to viralproteins, such as adenovirus particles, to facilitate endocytosis(Curiel et al., Proc. Natl. Acad. Sci. U.S.A. 88:8850-8854 (1991)). Inother embodiments, molecular conjugates of the instant invention caninclude microtubule inhibitors (WO/9406922), synthetic peptidesmimicking influenza virus hemagglutinin (Plank et al., J Biol. Chem.269:12918-12924 (1994)), and nuclear localization signals such as SV40 Tantigen (WO93/19768).

Retroviral vectors are also useful for introducing the nucleic acids ofthe invention into target cells or organisms. Retroviral vectors areproduced by genetically manipulating retroviruses. The viral genome ofretroviruses is RNA. Upon infection, this genomic RNA is reversetranscribed into a DNA copy which is integrated into the chromosomal DNAof transduced cells with a high degree of stability and efficiency. Theintegrated DNA copy is referred to as a provirus and is inherited bydaughter cells as is any other gene. The wild type retroviral genome andthe proviral DNA have three genes: the gag, the pol and the env genes,which are flanked by two long terminal repeat (LTR) sequences. The gaggene encodes the internal structural (nucleocapsid) proteins; the polgene encodes the RNA directed DNA polymerase (reverse transcriptase);and the env gene encodes viral envelope glycoproteins. The 5′ and 3′LTRs serve to promote transcription and polyadenylation of virion RNAs.Adjacent to the 5′ LTR are sequences necessary for reverse transcriptionof the genome (the tRNA primer binding site) and for efficientencapsulation of viral RNA into particles (the Psi site) (see, Mulligan,In: Experimental Manipulation of Gene Expression, Inouye (ed), 155-173(1983); Mann et al., Cell 33:153-159 (1983); Cone and Mulligan,Proceedings of the National Academy of Sciences, U.S.A.,81:6349-6353(1984)).

The design of retroviral vectors is well known to those of ordinaryskill in the art. In brief, if the sequences necessary for encapsidation(or packaging of retroviral RNA into infectious virions) are missingfrom the viral genome, the result is a cis acting defect which preventsencapsidation of genomic RNA. However, the resulting mutant is stillcapable of directing the synthesis of all virion proteins. Retroviralgenomes from which these sequences have been deleted, as well as celllines containing the mutant genome stably integrated into the chromosomeare well known in the art and are used to construct retroviral vectors.Preparation of retroviral vectors and their uses are described in manypublications including, e.g., European Patent Application EPA 0 178 220;U.S. Pat. No. 4,405,712, Gilboa Biotechniques 4:504-512 (1986); Mann etal., Cell 33:153-159 (1983); Cone and Mulligan Proc. Natl. Acad. Sci.USA 81:6349-6353 (1984); Eglitis et al. Biotechniques 6:608-614 (1988);Miller et al. Biotechniques 7:981-990 (1989); Miller (1992) supra;Mulligan (1993), supra; and WO 92/07943.

The retroviral vector particles are prepared by recombinantly insertingthe desired nucleotide sequence into a retrovirus vector and packagingthe vector with retroviral capsid proteins by use of a packaging cellline. The resultant retroviral vector particle is incapable ofreplication in the host cell but is capable of integrating into the hostcell genome as a proviral sequence containing the desired nucleotidesequence. As a result, the patient is capable of producing, for example,an Archipelin polypeptide of interest and thus restore the cells to anormal phenotype.

Packaging cell lines that are used to prepare the retroviral vectorparticles are typically recombinant mammalian tissue culture cell linesthat produce the necessary viral structural proteins required forpackaging, but which are incapable of producing infectious virions. Thedefective retroviral vectors that are used, on the other hand, lackthese structural genes but encode the remaining proteins necessary forpackaging. To prepare a packaging cell line, one can construct aninfectious clone of a desired retrovirus in which the packaging site hasbeen deleted. Cells comprising this construct will express allstructural viral proteins, but the introduced DNA will be incapable ofbeing packaged. Alternatively, packaging cell lines can be produced bytransforming a cell line with one or more expression plasmids encodingthe appropriate core and envelope proteins. In these cells, the gag,pol, and env genes can be derived from the same or differentretroviruses.

A number of packaging cell lines suitable for the present invention arealso available in the prior art. Examples of these cell lines includeCrip, GPE86, PA317 and PG13 (see Miller et al., J Virol. 65:2220-2224(1991)). Examples of other packaging cell lines are described in Coneand Mulligan Proceedings of the National Academy of Sciences, USA,81:6349-6353 (1984); Danos and Mulligan Proceedings of the NationalAcademy of Sciences, USA, 85:6460-6464 (1988); Eglitis et al. (1988),supra; and Miller (1990), supra.

Packaging cell lines capable of producing retroviral vector particleswith chimeric envelope proteins may be used. Alternatively, amphotropicor xenotropic envelope proteins, such as those produced by PA317 and GPXpackaging cell lines may be used to package the retroviral vectors.

In some embodiments of the invention, an antisense nucleic acid isadministered which hybridizes to a gene encoding an Archipelin of theinvention or to a transcript thereof. The antisense nucleic acid can beprovided as an antisense oligonucleotide (see, e.g., Murayama et al.,Antisense Nucleic Acid Drug Dev. 7:109-114 (1997)). Genes encoding anantisense nucleic acid can also be provided; such genes can beintroduced into cells by methods known to those of skill in the art. Forexample, one can introduce a gene that encodes an antisense nucleic acidin a viral vector, such as, for example, in hepatitis B virus (see,e.g., Ji et al., J Viral Hepat. 4:167-173 (1997)), in adeno-associatedvirus (see, e.g., Xiao et al., Brain Res. 756:76-83 (1997)), or in othersystems including, but not limited, to an HVJ (Sendai virus)-liposomegene delivery system (see, e.g., Kaneda et al., Ann. NY Acad. Sci.811:299-308 (1997)), a “peptide vector” (see, e.g., Vidal et al., CRAcad. Sci III 32:279-287 (1997)), as a gene in an episomal or plasmidvector (see, e.g., Cooper et al., Proc. Natl. Acad. Sci. U.S.A.94:6450-6455 (1997), Yew et al. Hum Gene Ther. 8:575-584 (1997)), as agene in a peptide-DNA aggregate (see, e.g, Niidome et al., J Biol. Chem.272:15307-15312 (1997)), as “naked DNA” (see, e.g., U.S. Pat. Nos.5,580,859 and 5,589,466), in lipidic vector systems (see, e.g., Lee etal., Crit Rev Ther Drug Carrier Syst. 14:173-206 (1997)), polymer coatedliposomes (U.S. Pat. Nos. 5,213,804 and 5,013,556), cationic liposomes(Epand et al., U.S. Pat. Nos. 5,283,185; 5,578,475; 5,279,833; and5,334,761), gas filled microspheres (U.S. Pat. No. 5,542,935),ligand-targeted encapsulated macromolecules (U.S. Pat. Nos. 5,108,921;5,521,291; 5,554,386; and 5,166,320).

C. Pharmaceutical Formulations

When used for pharmaceutical purposes, the vectors used for gene therapyare formulated in a suitable buffer, which can be any pharmaceuticallyacceptable buffer, such as phosphate buffered saline or sodiumphosphate/sodium sulfate, Tris buffer, glycine buffer, sterile water,and other buffers known to the ordinarily skilled artisan such as thosedescribed by Good et al. Biochemistry 5:467 (1966).

The compositions can additionally include a stabilizer, enhancer orother pharmaceutically acceptable carriers or vehicles. Apharmaceutically acceptable carrier can contain a physiologicallyacceptable compound that acts, for example, to stabilize the nucleicacids of the invention and any associated vector. A physiologicallyacceptable compound can include, for example, carbohydrates, such asglucose, sucrose or dextrans, antioxidants, such as ascorbic acid orglutathione, chelating agents, low molecular weight proteins or otherstabilizers or excipients. Other physiologically acceptable compoundsinclude wetting agents, emulsifying agents, dispersing agents orpreservatives, which are particularly useful for preventing the growthor action of microorganisms. Various preservatives are well known andinclude, for example, phenol and ascorbic acid. Examples of carriers,stabilizers or adjuvants can be found in Remington's PharmaceuticalSciences, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985).

D. Administration of Formulations

The formulations of the invention can be delivered to any tissue ororgan using any delivery method known to the ordinarily skilled artisan.In some embodiments of the invention, the nucleic acids of the inventionare formulated in mucosal, topical, and/or buccal formulations,particularly mucoadhesive gel and topical gel formulations. Exemplarypermeation enhancing compositions, polymer matrices, and mucoadhesivegel preparations for transdermal delivery are disclosed in U.S. Pat. No.5,346,701.

E. Methods of Treatment

The gene therapy formulations of the invention are typicallyadministered to a cell. The cell can be provided as part of a tissue,such as an epithelial membrane, or as an isolated cell, such as intissue culture. The cell can be provided in vivo, ex vivo, or in vitro.

The formulations can be introduced into the tissue of interest in vivoor ex vivo by a variety of methods. In some embodiments of theinvention, the nucleic acids of the invention are introduced into cellsby such methods as microinjection, calcium phosphate precipitation,liposome fusion, or biolistics. In further embodiments, the nucleicacids are taken up directly by the tissue of interest.

In some embodiments of the invention, the nucleic acids of the inventionare administered ex vivo to cells or tissues explanted from a patient,then returned to the patient. Examples of ex vivo administration oftherapeutic gene constructs include Nolta et al., Proc Natl. Acad. Sci.USA 93(6):2414-9 (1996); Koc et al., Seminars in Oncology 23 (1):46-65(1996); Raper et al., Annals of Surgery 223(2):116-26 (1996); Dalesandroet al., J Thorac. Cardi. Surg., 1 1(2):416-22 (1996); and Makarov etal., Proc. Natl. Acad. Sci. USA 93(l):402-6 (1996).

IX. ADMINISTRATION AND PHARMACEUTICAL COMPOSITIONS

Modulators of Archipelin (e.g., agonists, including Archipelinpolypeptides, and antagonists) can be administered directly to themammalian subject for modulation of Archipelin signaling in vivo.Administration is by any of the routes normally used for introducing amodulator compound into ultimate contact with the tissue to be treatedand well known to those of skill in the art. Although more than oneroute can be used to administer a particular composition, a particularroute can often provide a more immediate and more effective reactionthan another route.

The compounds of the present invention can also be used effectively incombination with one or more additional active agents depending on thedesired target therapy (see, e.g., Turner, N. et al. Prog. Drug Res.(1998) 51: 33-94; Haffner, S. Diabetes Care (1998) 21: 160-178; andDeFronzo, R. et al. (eds.), Diabetes Reviews (1997) Vol. 5 No. 4). Anumber of studies have investigated the benefits of combinationtherapies with oral agents (see, e.g., Mahler, R., J Clin. Endocrinol.Metab. (1999) 84: 1165-71; United Kingdom Prospective Diabetes StudyGroup: UKPDS 28, Diabetes Care (1998) 21: 87-92; Bardin, C. W.,(ed.),Current Therapy In Endocrinology And Metabolism, 6th Edition (Mosby YearBook, Inc., St. Louis, Mo. 1997); Chiasson, J. et al., Ann. Intern. Med.(1994) 121: 928-935; Coniff, R. et al., Clin. Ther. (1997) 19: 16-26;Coniff, R. et al., Am. J Med. (1995) 98: 443-451; and Iwamoto, Y. etal., Diabet. Med. (1996) 13 365-370; Kwiterovich, P. Am. J Cardiol(1998) 82(12A): 3U-17U). These studies indicate that modulation ofdiabetes and hyperlipidemia, among other diseases, can be furtherimproved by the addition of a second agent to the therapeutic regimen.Combination therapy includes administration of a single pharmaceuticaldosage formulation which contains an Archipelin modulator of theinvention and one or more additional active agents, as well asadministration of an Archipelin modulator and each active agent in itsown separate pharmaceutical dosage formulation. For example, anArchipelin modulator and a thiazolidinedione can be administered to thehuman subject together in a single oral dosage composition, such as atablet or capsule, or each agent can be administered in separate oraldosage formulations. Where separate dosage formulations are used, anArchipelin modulator and one or more additional active agents can beadministered at essentially the same time (i.e., concurrently), or atseparately staggered times (i.e., sequentially). Combination therapy isunderstood to include all these regimens.

Still another example of combination therapy can be seen in modulatingdiabetes (or treating diabetes and its related symptoms, complications,and disorders), wherein the AKR1 C modulators can be effectively used incombination with, for example, sulfonylureas (such as chlorpropamide,tolbutamide, acetohexamide, tolazamide, glyburide, gliclazide, glynase,glimepiride, and glipizide), biguanides (such as metformin), a PPAR betadelta agonist, a ligand or agonist of PPAR alpha such asthiazolidinediones (such as ciglitazone, pioglitazone (see, e.g., U.S.Pat. No. 6,218,409), troglitazone, and rosiglitazone (see, e.g., U.S.Pat. No. 5,859,037)); dehydroepiandrosterone (also referred to as DHEAor its conjugated sulphate ester, DHEA-SO4); antiglucocorticoids; TNFainhibitors; a-glucosidase inhibitors (such as acarbose, miglitol, andvoglibose), amylin and amylin derivatives (such as pramlintide, (see,also, U.S. Pat. Nos. 5,902,726; 5,124,314; 5,175,145 and 6,143,718,6,136,784)), insulin secretogogues (such as repaglinide, gliquidone, andnateglinide (see, also, U.S. Pat. Nos. 6,251,856; 6,251,865; 6,221,633;6,174,856)), insulin, as well as the active agents discussed above fortreating atherosclerosis.

The pharmaceutical compositions of the invention may comprise apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are determined in part by the particular composition beingadministered, as well as by the particular method used to administer thecomposition. Accordingly, there is a wide variety of suitableformulations of pharmaceutical compositions of the present invention(see, e.g., Remington's Pharmaceutical Sciences, 17^(th) ed. 1985)).

The modulators (e.g., agonists or antagonists) of the expression oractivity of the Archipelin , alone or in combination with other suitablecomponents, can be prepared for injection or for use in a pump device.Pump devices (also known as “insulin pumps”) are commonly used toadminister insulin to patients and therefore can be easily adapted toinclude compositions of the present invention. Manufacturers of insulinpumps include Animas, Disetronic and .MiniMed.

The modulators (e.g., agonists or antagonists) of the expression oractivity of the Archipelin, alone or in combination with other suitablecomponents, can be made into aerosol formulations (i.e., they can be“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for administration include aqueous and non-aqueoussolutions, isotonic sterile solutions, which can contain antioxidants,buffers, bacteriostats, and solutes that render the formulationisotonic, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. In the practice of this invention, compositions canbe administered, for example, orally, nasally, topically, intravenously,intraperitoneally, or intrathecally. The formulations of compounds canbe presented in unit-dose or multi-dose sealed containers, such asampoules and vials. Solutions and suspensions can be prepared fromsterile powders, granules, and tablets of the kind previously described.The modulators can also be administered as part a of prepared food ordrug.

The dose administered to a patient, in the context of the presentinvention should be sufficient to effect a beneficial response in thesubject over time. The optimal dose level for any patient will depend ona variety of factors including the efficacy of the specific modulatoremployed, the age, body weight, physical activity, and diet of thepatient, on a possible combination with other drugs, and on the severityof the case of diabetes. It is recommended that the daily dosage of themodulator be determined for each individual patient by those skilled inthe art in a similar way as for known insulin compositions. The size ofthe dose also will be determined by the existence, nature, and extent ofany adverse side-effects that accompany the administration of aparticular compound or vector in a particular subject.

In determining the effective amount of the modulator to be administereda physician may evaluate circulating plasma levels of the modulator,modulator toxicity, and the production of anti-modulator antibodies. Ingeneral, the dose equivalent of a modulator is from about 1 ng/kg to 10mg/kg for a typical subject.

For administration, Archipelin modulators of the present invention canbe administered at a rate determined by the LD-50 of the modulator, andthe side-effects of the inhibitor at various concentrations, as appliedto the mass and overall health of the subject. Administration can beaccomplished via single or divided doses.

X. DIAGNOSIS OF DIABETES

The present invention also provides methods of diagnosing diabetes or apredisposition of at least some of the pathologies of diabetes.Diagnosis involves determining the level of Archipelin in a patient andthen comparing the level to a baseline or range. Typically, the baselinevalue is representative of Archipelin in a healthy (i.e., non-diabetic)person. As discussed above, variation of levels (either high or low) ofArchipelin from the baseline range suggests that the patient is eitherdiabetic or at risk of developing at least some of the pathologies ofdiabetes. Variation van be, e.g., at least 5%, 10%, 20%, 50%, 200%,400%, 500%, or 1000% or more of a baseline value or range. In someembodiments, the level of Archipelin are measured by taking a bloodsample from a patient and measuring the amount of Archipelin in thesample using any number of detection methods, such as those discussedherein. For instance, fasting and fed blood or urine levels can betested.

Glucose tolerance tests can also be used to detect the effect of glucoselevels on Archipelin levels. In glucose tolerance tests, the patient'sability to tolerate a standard oral glucose load is evaluated byassessing serum and urine specimens for glucose levels. Blood samplesare taken before the glucose is ingested, glucose is given by mouth, andblood or urine glucose levels are tested at set intervals after glucoseingestion.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily 10 apparent to one of ordinary skillin the art in light of the teachings of this invention that certainchanges and modifications may be made thereto without departing from thespirit or scope of the appended claims.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1

This example describes the discovery of a new member of theCRF/urocortin family of peptides that is also highly abundant inpancreatic islets and is a hormone important for the treatment ofdiabetes mellitus.

The probe set MBXRATISLl2276 was identified as a gene specificallyexpressed in rat islets using rat tissue map experiments on rat isletGeneChips. Analysis of a second, more extensive, rat tissue mappingexperiment also revealed that probe set MBXRATISL12276 was abundant inrat islet samples and absent in 10 samples from 10 other tissues. Thisprobe set was present in 5 out of 5 rat islet samples with an averageAverage Difference of >4000 and was absent in all 10 other tissues withan average Average Difference value of 0 or less. This represents anextremely islet specific expression pattern. See FIG. 1. A mouse tissuemap experiment also showed an extremely islet specific expressionpattern. See FIG. 2. The predicted product of the gene corresponding tothese probe sets was designated Archipelin.

Expression of Archipelin in diabetic animal models was also determined.This data indicated that probe set MBXRATISL12276 expression is 3-foldlower in Zucker Diabetic Fatty male rats versus ZLC control rats. Thisprobe set is also lower in diabetic (fat fed) ZDF female rats. This dataindicates that this gene is down-regulated in the islets of these animalmodels of Type 2 diabetes. These findings indicate that pancreaticislets are the major site of Archipelin production and suggests thatthis hormone is inappropriately expressed in individuals with diabetesmellitus. Type 1 diabetes is characterized by destruction of islets, andthis will very likely lead to Archipelin levels to be reduced or absent.Islet disfunction is also a critical aspect of Type 2 diabetes andtherefore also effects Archipelin production.

The nucleotide sequence of the mouse, rat and human Archipelin cloneswere determined. SEQ ID NO: 1 and SEQ ID NO:8 display two variants ofthe human sequence, SEQ ID NO:3 shows the mouse sequence and SEQ ID NO:5shows the rat sequence. The predicted amino acid sequences ofunprocessed Archipelin are shown for human (SEQ ID NOs:2 and 7), mouse(SEQ ID NO:4) and rat (SEQ ID NO:6). Depending on the human clonesequenced, amino acid 94 was either arginine or glycine. Multiple clonesencoded each amino acid.

Using MBXRATISL12276 to BLAST search a human islet endocrine celldatabase revealed ortholog clones among the human islet ESTs. CDNAclones for Archipelin were also abundant in the mouse and rat islet estdatabases. Subsequent Northern blot hybridizations with both rat andhuman cDNA clones of Archipelin confirm the islet specific expression ofan approximately 1.2 kb Archipelin transcript.

A BLAST search of the public databases revealed significant similarityto swissprot+ database entry Q9i8e5 (fugu rubripes (japanese pufferfish)(takifugu rubripes)), which was designated as thefugu urocortinprecursor. Further alignments suggested that the Archipelin peptide wasalso related to Corticotropin Releasing Factor (CRF) family. CRF is akey regulatory component of the hypothalamus-pituitary-adrenal axis.See, FIG. 3 illustrating an alignment of Archipelin amino acid sequenceswith members of the CRF peptide family. In light of the sequenceanalysis, it was determined that the Archipelin peptide was a processed,secreted peptide. FIGS. 3-5 illustrate the possible processing sites ofthe human, mouse and rat Archipelin peptides, respectively.

As noted above, the predicted Archipelin peptide sequence has a CRFfamily signature as well as a C-terminal proteolytic processing sequence(glycine-basic-basic-basic) that is characteristic for this family andresults in C-terminal amidation. In the case of Archipelin from mouse,rat and human, this will result in an isoleucyl-amide. There are alsobasic residues in the region that is expected to be an N-terminalprocessing site (FIGS. 4-6) producing a mature peptide containingbetween 50 and 38 amino acid residues. More than one of these may existphysiologically and one or all of these may be biologically active. Itis also possible that a larger version containing some or all of thepropeptide has biological function.

A predicted mature peptide sequence for rat Archipelin(TKFTLSLDVPTNIMNILFNIDKAKNLRAKAAANAQLMAQI-CONH2; SEQ ID NO:41) wasobtained and used in a variety of biological studies. Without intendingto limit the scope of the invention, it is predicted that this peptiderepresents a likely mature, processed version of the peptide.

In addition, the peptide sequence Cys-LFNIDKAKNLRAK (SEQ ID NO:54),which represents residues 137 to 149 of the coding sequence of both ratand mouse Archipelin precursors was used to develop anti-sera. Anaffinity-purified version of this antibody recognizes the in vitrotranslated 160 amino acid rat Archipelin precursor molecule. Thisantiserum also recognizes the Archipelin precursor expressed bytransfection of the rat Archipelin cDNA in HEK 293 cells on a westernblot, as well as the 40-mer form of Archipelin(TKFTLSLDVPTNIMNILFNIDKAKNLRAKAAANAQLMAQI-CONH2; SEQ ID NO:41).

Immunohistochemical studies using these antibodies demonstrated thatArchipelin is highly expressed in islet cells, and in particular, inβ-cells.

Moreover, in diabetes animal model systems, the animals typicallydisplayed reduced levels of Archipelin their blood before a drop ininsulin levels. These results indicate that monitoring Archipelin levelsis particularly useful as an early indicator of a predisposition fordiabetes.

Archipelin in Human Serum Samples

Human archipelin peptide was detected in normal human serum samplesusing surface enhanced laser desorption/ionization (SELDI) massspectroscopy of peptides specifically captured on a surface coated withaffinity-purified anti-archipelin antibody (FIG. 7). This wasaccomplished using a Ciphergen protein chip reader instrument (SeriesPBS II). Affinity purified antibody was covalently bound to a PS20pre-activated chip surface for the affinity capture of archipelin.Antibody was incubated on the chip surface overnight at 2-8° C. The 8spot chip was then blocked for 30 minutes using a free amine from a IMsolution of ethanolamine. Dilutions (1:70) of human serum in PBScontaining 0.05% Triton X were then applied to each of the spots andincubated for two hours at room temperature. Non-specific proteins wereremoved through post binding washes in PBS containing 0.05% triton-X.The energy-absorbing molecule CHCA was applied to the chip surface toform a crystal surface. Once crystal formation occurred the capturedprotein was ionized and read with the protein chip reader. The resultsrevealed highly reproducible peaks at masses of 4472-4476 (monomer) and8932-8936 (dimer). This size is consistent with the major serum form ofthe archipelin peptide being 40 or 41 amino acids(TKFTLSLDVPTNIMNLLFNIAKAKNLRAQAAANAHLMAQI (SEQ ID NO:55) orRTKFTLSLDVPTNIMNLLFNIAKAKNLRAQAAANAHLMAQI (SEQ ID NO:56)).

Trypsin Digestion Pattern of Rat Archipelin

After antibody capture of archipelin from rat serum using a PS 20pre-activated Ciphergen chip, the peptide was digested using modifiedsequencing grade trypsin. Resulting peaks were identified based onpredicted sequence from peptide cutter (expasy.org) (FIG. 8). The massof the thirty-eight amino acid peptide of rat archipelin from rat was4172 daltons. Capture of the native form of archipelin from serum wasapproximately 4150 daltons, which is within 0.5% of the expected mass.The major serum form of rat archipelin was therefore the 38-mer(FTLSLDVPTNIMNILFNIDKAKNLRAKAAANAQLMAQI; SEQ ID NO:57). Predictedtrypsin digestion products closely matched the observed archipelindigestion.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. An isolated polypeptide comprising SEQ ID NO:2.